ACTA POLONIAE PHARMACEUTICA - ptf.content-manager.plptf.content-manager.pl/pub/File/Acta_Poloniae/2016/nr 6/acta6_2016.pdf · Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73

Drug Research

ACTA POLONIAEPHARMACEUTICAVOL. 7��3� No. �6� ��N�ovember/December 201�6 � � � � � � ISSN 2353-5288

EDITOR

Aleksander P. MazurekNational Medicines Institute, The Medical University of Warsaw

ASSISTANT EDITOR

Jacek BojarskiMedical College, Jagiellonian University, KrakÛw

EXECUTIVE EDITORIAL BOARDThe Medical University of Warsaw

The Medical University of GdaÒsk

The Medical University of Warsaw

K. Marcinkowski University of Medical Sciences, PoznaÒ

The Medical University of Wroc≥aw

Polish Pharmaceutical Society, Warsaw

Czech Pharmaceutical Society

Charles Sturt University, Sydney

Pharmazeutisches Institut der Universit‰t, Bonn

DOV Pharmaceutical, Inc.

Semmelweis University of Medicine, Budapest

Boøenna GutkowskaRoman KaliszanJan PacheckaJan PawlaczykJanusz PlutaWitold WieniawskiPavel KomarekHenry Ostrowski-MeissnerErhard RˆderPhil SkolnickZolt·n Vincze

This Journal is published bimonthly by the Polish Pharmaceutical Society (Issued since 1937)

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Acta Poloniae Pharmaceutica ñ Drug Research

Volume 73, Number 6 November/December 2016

CONTENTS

REVIEW

1415. Muhammad Zaman, Syed Saeed-ul-Hassan, Pellets and pelletization: emerging trends in the pharma industry.Rai Muhammad Sarfraz, Nighat Batool, Muhammad Junaid Qureshi, Muhammad Abdullah Akram, Saiqa Munir, Zeeshan Danish

1427. Beata M. Gruber-Bzura Vitamin D in Alzheimerís desease - prophylaxis or therapy?

1433. Ewelina Dziwota, Marcin Olajossy Vortioxetine ñ the new antidepressant agent with procognitive properties.

ANALYSIS

1439. Pawe≥ Ramos, Sylwia Jarco, Piotr PepliÒski, Free radical formation in rosuvastatin during thermal sterilization Barbara Pilawa at different temperatures.

1447. Eløbieta KrÛlak, Jolanta Raczuk, Lidia Borkowska Trace metal content (Cu, Zn Mn, and Fe) in Urtica dioica L. and Plantago major L.

1455. Tomasz Wasy≥yszyn, Katarzyna Borowska Studies on stability of 2,3-diphenylcyclopropenone in contact with water and aqueous NaCl solutions. Conclusions for purpose of topical therapy of patients with alopecia areata.

1461. Marina Z. Zekic Stosic, Sandra M. Jaksic, Igor M. Optimization and validation of HPLC method for tetramethrin Stojanov, Jelena B. Apic, Radomir D. Ratajac determination in human shampoo formulation.

1467. Krystyna CzerwiÒska, Eløbieta Wyszomirska, Eløbieta Identification and determination of rupatadine and fexofenadine Kublin, Ewa Malanowicz Aleksander P. Mazurek by densitometric method.

1475. Anna Petruczynik, Karol WrÛblewski, Szymon StrÛøek, RP-HPLC analysis of acidic and basic drugs in systems with Monika Waksmundzka-Hajnos diethylamine as eluents additive.

1487. Katarzyna Buú-Kwaúnik, Monika Filist, Piotr J. Rudzki Environmentally friendly LC/MS determination of eplerone in human plasma.

1495. Micha≥ Kaza, Edyta PiÛrkowska, Monika Filist, HPLC-UV assay of imatinib in human plasma optimized for Piotr J. Rudzki bioequivalence studies.

DRUG BIOCHEMISTRY

1505. Renata Francik, Natalia Szkaradek, Dorota Øelaszczyk, Antioxidant activity of xanthone derivatives.Henryk Marona

1511. Cecylia £ukaszuk, Eløbieta Krajewska-Ku≥ak, Andrzej In vitro antifungal activity of 2,4-dihydroxy-N-(3-thioxo-Niewiadomy, Beata Szynaka 3H-1,2,4-dithiazol-5-yl) benzenecarbothioamide.

1521. Ping Wang, Bin Shi, Zhi-Hui Gao, Tie-Feng Sun, Effect of Colla Cornus Cervi combined with LV-mediated Wu-Bin Yang, Shu-Fang Han, Peng Liu, Lei-Lei Wang, BMP7 transfected BMSCs on ANFH in rats. Bo-Nian Zhao, Dan-Dan Wang

1531. Anna Bilska-Wilkosz, Magdalena GÛrny, Magdalena Inactivation of aldehyde dehydrogenase by nitroglycerin in Dudek, Leszek NowiÒski, Marek Bednarski, Ma≥gorzata the presence and absence of lipoic acid and dihydrolipoic acid. Iciek, Danuta Kowalczyk-Pachel, Maria Implications for the problem of differential effects of Soko≥owska-Jeøewicz, Barbara Filipek, Lidia W≥odek lipoic acid in vitro and in vivo

1539. Marek Juda, Pawe≥ Helon, Anna Malm Anti-adhesive and anti-biofilm activities in vitro of linezolid, vancomycin, tigecycline and daptomycin against Staphylococcus haemolyticus.

APPHAX 73 (6) 1413 ñ 1670 (2016)

DRUG SYNTHESIS

1545. Anna Czopek, Agnieszka ZagÛrska, Marcin Ko≥aczkowski, New spirohydantoin derivatives - synthesis, pharmacological Adam Bucki, Beata Gryz≥o, Joanna Rychtyk, Maciej evaluation and molecular modeling study.Paw≥owski, Agata Siwek, Grzegorz Sata≥a, Andrzej Bojarski, Monika Kubacka, Barbara Filipek

NATURAL DRUGS

1555. Muniba Jadoon, Nighat Fatima, Sidra Murtaza, Production of antimicrobial peptides by Epicoccum sp. Leng Chee Chang, Naeem Ali, Safia Ahmed NFW1: an endophyte of Taxus fauna.

1565. Ivana D. RadojeviÊ, Sava M. VasiÊ, Milan S. DekiÊ, Antimicrobial and antibiofilm effects of extracts from Niko S. RaduloviÊ, Gorica T. –eliÊ, Jelena S. –ur�eviÊ, Trapa natans L., evaluation of total phenolic and Ljiljana R. »omiÊ flavonoid contents, and GC-MS analysis.

1575. Shafqat Rasul Chaudhry, Adnan Akram, Naveed Aslam, Antidiabetic and antidyslipidemic effects of Heliotropium Muhammad Asif, Muhammad Wajid, Thomas Kinfe, strigosum in rat models of type I and type II diabetes.Qaiser Jabeen, Sajjad Muhammad

1587. Hesham R. El-Seedi, Mohamed Gomaa, Mousa Maali Cytotoxic effects of the Red Sea soft coral Sacrophyton Salem, Hager M. Krshk, Nermeen Yosri, Ahmed Ayesh, trochliophorum. Ahmed Askar, Kawither Soliman, Zeinab Hamza, Hager M. Mansour, Ahmed Elkhatheb, Shaden A.M. Khalifa

1593. Olumuyiwa O. Ogunlaja, Roshila Moodley, Himansu Chemical constituents and in vitro antioxidant activity of Baijnath, Sreekantha B. Jonnalagadda crude extracts and compounds from leaves, and stem bark of

Ficus burtt-davyi.

1601. Amir Mehmood, Irfan Hamid, Ali Sharif, Muhammad Evaluation of anti-inflammatory, analgesic and antipyretic Furqan Akhtar, Bushra Akhtar, Ammara Saleem, activities of aqueous and ethanolic extracts of seeds of Javed Iqbal, Maryam Shabbir, Sajid Ali Buchanania lanzan Spreng. in animal models.

1609. Beata Olas, Bogdan Kontek, Wieslaw Oleszek, Commercial extract from aronia as a modulator of adhesive Anna Stochmal properties of fibrinogen treated with homocysteine and its

thiolactone in vitro.

1615. Simin Babaeizadeh, Saeed Heydarnejhad, Abdollah Wound healing activity of extract from Thymus daenensisGhasemi Pirbalouti, Faham Khamesipoor, Elham in burn wound model: an experimental animal study.Moghtadaei-Khorasgani, Parisa Heydari-Soureshjani

PHARMACEUTICAL TECHNOLOGY

1623. Lina Babickait��, Kristina Ramanauskien�

�, Aidas Grigonis, Determination of antimicrobial activity of chlorhexidine gel.

Marija Iva�kien��, Gintaras Daunoras, Irena Klimien�

�,

Marius Virgailis, Gintaras Zamokas, Asta Marija Ink��nien�

�,

Algimantas Petras MatuseviËius

1631. Timucin Ugurlu, Aysun Nalbantoglu, Ceyda Development of solid lipid nanocarriers for oral delivery of Tuba Sengel-Turk candesertan cilexetil.

1639. Ayesha Yaqoob, Mahmood Ahmad, Asif Mahmood, Preparation, in vitro and in vivo characterization of hydrophobic Rai Muhammad Sarfraz patches of a highly water soluble drug for prolonged plasma half

life: effect of permeation enhancers.

PHARMACOLOGY

1649. Jing Lu, Shenggang Wang, Guiying Chen, Xiaofeng Sun, The investigation of effect of flurbiprofen axetil on the tissue Kezhong Li growth and the content of PGE2 in cervical cancer.

GENERAL

1653. Orsolya Somogyi, Rom·na ZelkÛ Patientsí knowledge of medical patches in Hungary.

1659. Aubid Ullah Khan, Naveed Akhtar, Asadullah Madni, Socio-economic constrains faced by tuberculosis patients that lead Nayab Tahir, Mubashar Rehman, Ahmed Raza to non-compliance - a cross sectional study in Southern Punjab,

Pakistan.

SHORT COMMUNICATION

1665. Sadia Chuman, Hammad Saleem, M Shoaib Ali Gill, Evaluation of spermicidal property of Hippophae rhamnoides L. Nabeel Shahid, Fizza Karamat, Tabish Ali, Faisal berries. Nadeem, Waqas Mehmood

1669. Erratum

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 6 pp. 1415ñ1425, 2016 ISSN 0001-6837Polish Pharmaceutical Society

Most widely used route of administration is theoral route. Oral drug administration has been one ofthe most convenient and widely accepted routes ofdelivery for most therapeutic agents. Pellets aredefined as the small sized agglomerates of granulesor powders having spherical shape with free flowingproperties, prepared using suitable pelletizationtechnique (1). Pellets are made of multiunit soliddosage form, which can be compressed in tablets,filled in capsules or may be suspended in a suspen-sion. Due to added benefits of their multiunit sys-tem, they are preferred over ordinary solid dosageforms (2). The diameter of the pellets ranges from0.5 to 1.5 mm that can be easily coated with suitablecoating material (3). It was in 1950s when the firstpellet formulation was introduced in the market (4).Pelletization techniques are playing leading rolefrom the last two decades in the field of pharmaceu-

tical drug delivery system. Pellets are formulatedmostly as modified release oral dosage form withsustained effect (5). Different techniques of pelleti-zation are invented. Layering processes have beenused over the years. Those processes have some lim-itations such as non-uniformity in the size of the pel-lets and less drug loading. In recent years, extrusion-spheronization, cryopelletization, freeze pelletiza-tion and hot melt extrusion have been used to pro-duce spherical pellets. The purpose of this review isto provide some information about the techniquesused for the manufacturing of pellets and polymersused for their formulations.

Pellets

Pellets are small free flowing, spherical dosageforms that are prepared by the agglomeration of finepowder mixture of drug and excipients (5, 6). Pellets

REVIEW

PELLETS AND PELLETIZATION: EMERGING TRENDS IN THE PHARMA INDUSTRY

MUHAMMAD ZAMAN1,2*, SYED SAEED-UL-HASSAN1, RAI MUHAMMAD SARFRAZ3, NIGHAT BATOOL1, MUHAMMAD JUNAID QURESHI2, MUHAMMAD ABDULLAH AKRAM3,

SAIQA MUNIR1 and ZEESHAN DANISH4

1Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan2Department of Pharmacy, Bahauddin Zakariya University, Multan 60800, Pakistan

3 Faculty of Pharmacy, University of Sargodha, Pakistan4University College of Pharmacy, Punjab University, Lahore, Pakistan

Abstract: The present time is considered as an era of advancements in drug delivery systems. Different novelapproaches are under investigation that range from uniparticulate to multi particulate system, macro to microand nano particulate systems. Pelletization is one of the novel drug delivery technique that provides an effec-tive way to deliver the drug in modified pattern. It is advantageous in providing site specific delivery of thedrug. Drugs with unpleasant taste, poor bioavailability and short biological half-life can be delivered efficient-ly through pellets. Their reduced size makes them more valuable as compared to the conventional drug deliv-ery system. Different techniques are used to fabricate the pellets such as extrusion and spheronization, hot meltextrusion, powder layering, suspension or solution layering, freeze pelletization and pelletization by direct com-pression method. Various natural polymers including xanthan gum, guar gum, tragacanth and gum acacia, semi-synthetic polymers like cellulose derivatives, synthetic polymers like derivatives of acrylamides, can be used inpellets formulation. Information provided in this review is collected from various national and internationalresearch articles, review articles and literature available in the books. The purpose of the current review is todiscuss pellets, their characterizations, different techniques of pelletization and the polymers with potential ofbeing suitable for pellets formulation.

Keywords: pellets, pelletization, pelletizer, polymers, novel drug delivery system

1415

* Corresponding author: e-mail: [email protected]

1416 MUHAMMAD ZAMAN et al.

as drug delivery systems provide not only techno-logical advantages including better flow properties,durable dosage form, reduced particle size distribu-tion, simplicity of coating, and homogeneous pack-ing but also provide therapeutic advantages such ascomparatively less irritation of the alimentary canal,a lower danger of side effects related with dosedumping and a uniform distribution in the gastroin-testinal tract resulting in a decreased peak plasmafluctuations.

Desired characteristics of pelletsPellets should be of spherical shape and

smooth surface to achieve good flow characteristics,with particle size, preferably in the range of 600-1000 µm. They should have maximum drug loadingcapacity to maintain the desired size of the pellets.

Advantages and disadvantages of pellets (2, 4, 6, 9)Pelletization of solid oral dosage forms is com-

monly done either to deliver the drug at a particular

Table 1. Suitable properties of the drugs to be formulated in pellets (39).

Serial No. Nature of the properties

Drugs having low molecular weightDrugs with good water solubility pH independent With non-aqueous solubility

Physicochemical Unionized (at least 0.1-5%) in GI tract 1properties Very weak bases pKa < 5.0

Very weak acids pKa > 8.0 (Pentobarbital pKa = 8.1) Unionized at all pH Moderately weak acids pKa 2.5-5 Moderately weak bases

2Pharmacokinetic Short half-lifeproperties Well absorbed from all regions of GI tract

3 Pharmacodynamic Therapeutic range of blood concentration -property wide enough

Table 2. Drugs being used in pellets.

No. Drug name References

1 Ascorbic acid (40)

2 Aceclofenac (41)

3 Atenolol (42)

4 Baclofen (43)

5 Chlorpheniramine maleate (44)

6 Diltiazem HCl (45)

7 Duloxetine HCl (46)

8 Fluoxetine (47)

9 Lansoprazole (48)

10 Lornoxicam (49)

11 Metoclopramide (35)

12 Omeprazole (50)

13 Phenylephrine HCl (51)

14 Quetiapine fumarate (52)

15 Sertraline HCl (53)

16 Tramadol HCl (54)

17 Venlafaxine HCl (55, 56)

Pellets and pelletization: emerging trends in the pharma industry 1417

site within the gastrointestinal tract or to modify therelease and action of drugs. Pelletization techniquesare used sometimes to enhance the pharmaceuticalelegance of dosage form. Pelletization providesvariety in the development of formulation anddosage form design. It improves the flow propertiesof the formulations. In case of capsule filling,enhanced flow properties of the pellets helps to pro-vide uniform filling with reproducible filled weightof the capsules. One of the most important benefitsof pelletization is that it discourages the dose dump-ing and in this way, it can enhance both safety andeffectiveness of the drugs.

Resistance to agglomeration is beneficial forthe drug that can cause irritation. Pelletization pro-vides a suitable way for separation of incompatibledrugs in a dosage form.

Pellets have little effect on gastric emptyingtime and intestinal transit time. They provide freeand easy dispersion in gastrointestinal tract withsuitable absorption rate and decreased level of vari-ation in plasma concentration.

Taste masking can also be done efficientlythrough pelletization. Pelletization also provides anease of coating different drugs over the pellets withdesired release rate. Larger surface area of the pel-lets makes them suitable for rapid release formula-tions. In spite of these advantages, pellets also have

few disadvantages like - pellets are rigid and so can-not be compressed into tablets easily. Pellets pro-duction process is difficult and requires expensiveand special equipment.

Drugs suitable for pellets dosage form

Different drugs can be incorporated in pelletsfor their administration. Properties of suitable candi-dates are given in Table 1. Few examples of thedrugs that are used in pellets using different pelleti-zation techniques are listed in Table 2.

Drugs marketed as pellets

Pellets is an emerging trend in the pharmaceu-tical industry and researchers are working to putthem from research laboratories to the market. Fewexamples of drugs that are marketed as pelletsdosage from are listed in Table 4.

Polymers used in pellets formulation

Polymers have a significant impact as well aseffect in the pharmaceutical industry. Polymers canbe used in various types of drug delivery systemincluding gastro retentive drug delivery system,matrix tablets, buccal films, hydrogels and mucoad-hesive drug delivery system (10-14). A wide rangeof polymers is available that can be used in the for-mulation of pellets. Natural, semi synthetic as well

Table 3. List of polymers used in pelletization.

No. Name of the polymer References

1 Cellulose diacetate (52)

2 Cellulose acetate phthalate (57)

3 Chitosan (58)

4 Carboxymethyl cellulose (59)

5 Eudragit L30 (54)

6 Eudragit RS100 (49)

7 Ethyl cellulose (52)

8 Hypromellose phthalate (52)

9 Hypromellose acetate succinate (46)

10 Hydroxypropyl methyl cellulose (50)

11 Microcrystalline cellulose (56)

12 Polyvinyl acetate phthalate (46)

13 Polymethacrylic acid copolymer (46)

14 Polyvinylpyrrolidone (40, 60)

15 Sodium alginate (61, 62)

17 Sodium carboxymethyl cellulose (57)

18 Guar gum (63)

19 Xanthan gum (49)


as synthetic polymers can be used in the pelletiza-tion. Few of these polymers are listed in Figure 1and Table 3.

Pelletization techniques (15)Various techniques have been used for pelleti-

zation (Fig. 2).● Pelletization by:● Extrusion spheronization● Drug layering● Dry powder layering● Solution and suspension layering● Direct compression● Cryopelletization● Hot melt extrusion● Balling● Freeze pelletization

Extrusion spheronization (15, 16)This technique is used in the pharmaceutical

industry for the formulation of spherical particles ofeven size. It is useful technique for the preparationof pellets/granules with potential of high drug load-ing capacity. The formulated pellets can be used for

the development of sustained released oral dosageforms. This technique has the benefit of minimumpossible use of excipients, simple, easy and fast pro-cessing and high efficiency. It is a multi-step processas described below:

Dry mixing

This step is used to achieve uniform mixing ordispersion of dry powder. A variety of mixers areavailable that can be used for this purpose like twinshell, high shear, planetary and tumbler mixers (17).

Wet massing

This process is comparable to wet granulationtechnique that is used to produce granules but wetmassing in pelletization is used to prepare suitableplastic mass for extrusion. Commonly availablemixtures are planetary mixer, sigma blade mixer,high shear mixer and Horbat mixer (17).

Extrusion

This is the most important step of pelletizationin which pressure is applied to prepared mass to passit through the opening of extruder of desired dimen-

Table 4. Examples of drugs marketed as pellets.

No. Drug Manufacturer Product Therapeutic class

1 Omeprazole magnesium Astra Zeneca Losec MUPS Antiulcer

2 Esomeprazole magnesium Astra Zeneca Esomeprazole Antiulcer

3 Metoprolol tartrate Astra Zeneca Toprol XL Antihypertensive

4 Lansoprazole Takeda Prevacid solu Tab Antiulcer

5 Theophylline Key Theodur Antiasthmatic

Figure 1. Flow chart of pelletization techniques


sions. Rod shaped extrudes are prepared with suit-able plasticity. Plasticity should be sufficient toresist the deformation of extrudes but not enough tosupport the adhesion of the agglomerates. The sol-vent used in pelletization is responsible for bindingas well as lubrication to facilitate the extrusionprocess (18).

Spheronization

Rod shaped agglomerates are spheronized inthis process. Extrudes are placed in the spheronizerand rotated at high speed by friction plate that con-vert them to small sized spherical particles (19).

Drying (20)This process is performed to achieve required

level of moisture contents in the formulations.Pellets can be dried at room temperature and even athigher temperature if required. Freeze drying tech-nique, tray drying and fluidized bed drying tech-niques are available for drying of pellets. Freezedrying technique has the advantage over other tech-niques that it not only maintains the shape of the pel-lets but also retains the size (17).

Screening

Desired size of the pellets with uniform distri-bution is necessary and it is usually achieved by thesimple technique of sieving. Depending upon the

method of feed and transfer of the feed mass, dif-ferent extruders are available that are screw fed,gravity fed, ram extruders and marumerizers (17,20).

Screw fed extruders

In the screw fed extruders mass is transportedhorizontally with the help of screw that rotates alongthe horizontal axis. They also categorized as:

Axial screw extrudersIt contains a zone for feeding, a compression

zone and extrusion zone. Die plate is placed axially.

Radial screw extrudersIn radial screw extruders the transport zone is

comparatively short that cause immediate extrusionthrough mounted screen. The screen is placedaround the horizontal axis of the screws.

Gravity-fed extruders

Gravity fed extruders is further categorized as:

The rotary cylinderIt consists of two cylinders; one is hollow

cylinder while other is solid and acts as pressurecylinder. Both these cylinders perform counter rota-tion.

Figure 2. Principle of spheronization process

Figure 3. Various types of extruders


Rotary-gear extruderIt contains two hollow cylinders with counter

rotation and mounted with counter bored holes.

Ram extruders

It is one of the oldest types of extruder. Usuallyit is used in the development phase of the productsand also to evaluate the rheological properties. Itcontains a piston that displaces and pushes the mate-rial to pass through the screen.

Marumerizer

It contains a static cylinder and a rotating fric-tion plate. Friction plate has variable rotation speedthat ranges from 100-2000 rpm that depends on thediameter of the working unit.

Although the extrusion/spheronization is aneffective technique which is widely used in thepreparation of pellets, but it is an expensive and timeconsuming process as discussed above that is a mul-tistep process that requires a lot of time for the com-pletion.

Drug layering

It is the technique of pelletization which isused to coat or layer the seed material in powder,solution or suspension form. Initially, drug solutionor suspension is prepared by using suitable solventor non-solvent, respectively. Prepared solution or

suspension of the drug is then sprayed over the inertspherical core, usually prepared by using microcrys-talline cellulose or sugar. This process results in theformulation of varying sized pellets with inner coreand outer shell having different composition. Thistechnique has different types including: dry powderlayering, solution and suspension layering and directpelletization (16, 21).

Dry powder layering

This is the process in which consecutive lay-ers of dry powder of the drug or/and excipients aredeposited on preformed core. First, a binding solu-tion is prepared having a suitable binder. The pre-pared binding solution is sprayed over the inertcore of microcrystalline cellulose or sugar to pre-pare a sticky core with the ability to bind the drugpowder over it. The drug, which is to be layeredover prepared core, is grinded or micronized, ifrequired, to prepared fine powder and then thefinally divided powder is sprinkled over the inertspherical core in controlled manner to achieve uni-form sized circular pallets (22, 23). Conventionalcoating pan is usually used for this purpose but ithas few drawbacks like poor mixing and poor dry-ing. The substrate particles are fluidized and sus-pended by heated and conditioned air. One or sev-eral nozzles atomize and spray the drug powderonto the substrate.

Figure 4. Principle of powder layering

Figure 5. Principle of solution layering


Suspension / solution layering

In this process, successive layering ofsolution/suspension of ingredient including binder onstarter seeds is done. Starter seeds are usually of inertmaterial or may be of the same drug. In this method,solution or suspension of active ingredients along withother excipients is prepared. This solution/suspensionis sprayed over the core material. Fluid-bed apparatus,traditionally a Wurster column (Wurster HS, Glatt). Inthe Wurster column, the substrate particles are flu-idized and suspended by heated and conditioned air.One or several nozzles atomize and spray the drug dis-persion onto the substrate. The heated and conditionedair then evaporates the liquid carrier, leaving the drugdeposited on the substrate. Drying process is veryimportant as it crystallizes the dissolved material thatlink the core with consecutive layers of the drug orother polymers. This process is continued until therequired drug or polymers layer is achieved (2, 24).

It is also an effective technique but there arefew drawbacks of this and one of them is difficultyin achieving evenness in the drug distribution anduniformity in the size of the pellets.

Direct pelletization (24)In this technique homogeneous pellets with uni-

form particle size distribution can be prepared. Allexcipients are mixed homogenously with asolvent/binder which is then centrifuged. Centrifugal

force acts to form lumps which are then spheronizedto the pellets. Characteristics of pellets depend uponrotation speed, but in this method of pelletization, it isdifficult to identify the core. Usually, high shearmixer and fluidized bed apparatus is used for the dry-ing of pellets formed through direct pelletization (25).

Cryopelletization

It is a unique process as it requires a fixingmedium. Usually liquid nitrogen is used as fixingmedium which is applied on the droplets of liquidformulation to convert them into solid pellets. Thistechnique is similar to the one which is used for thelyophilization of viscous bacterial suspension.Liquid nitrogen prepares pellets at the temperatureof 160OC which causes vigorous transformation ofheat between drugs loaded droplets and fixing medi-um. Amount of solid and temperature of the solutionor suspension describe how much nitrogen shouldbe used in this process. Apparatus for this processconsists of a perforated plate, a conveyor belt whichacts as a reservoir, transport baffles storage contain-er. Droplets are generated through perforated platesand freeze when exposed to liquid nitrogen andfinally extracted out from the medium and stored at-160OC (25, 26).

It is a unique method for the preparation whichis an expensive one and required expertise for thepreparation of pellets by using this technique.

Figure 6. Principle of suspension layering

Figure 7. Principle of direct pellitization


Hot melt extrusion

Hot-melt extrusion is one of the most exten-sively used techniques in different industries likeplastic, rubber and food industry. Nowadays, thistechnique is succeeded in gaining entry into thepharmaceutical industry. In pharmaceutical indus-try, this technique is valuable in formulating differ-ent dosage forms including pellets, granules andtransdermal drug delivery systems (27, 28). In thismethod, drugs and other excipients are mixed even-ly and then melted on a temperature which is highenough to convert the ingredients in molten state.Usually a spheronizer is used to convert the pelletsinto spherical form (25, 28).

It is a very effective and used full method forpelletization but heat labile drugs and excipientscannot be fabricated as pellets.

Balling

It is method of pelletization which requires theaddition of sufficient quantity of liquid or exposureof the powder to high temperature during pelletiza-tion. Continuous tumbling or rolling action convertthe powder in to spherical aggregates (23). It isdivided into two types;

Liquid based agglomerationMelt based agglomeration

Figure 10. Freeze pelletizer I and II

Figure 8. Principle of cryopelletization Figure 9. Principle of hot melt extrusion


Liquid based agglomeration In liquid based agglomeration method, a liquid

is added to the powder. Liquid can be added beforeor during agitation. When the powder materialcomes in contact with liquid, it is converted intoagglomerates. Liquid phase may contain a harden-ing and binding agent that causes the binding of theagglomerates with adjacent ones. The agglomeratescollide with other agglomerates and form largeragglomerates or pellets. At this stage, layeringreplaces the coalescence and smaller particle stickon much larger particles that cause an increase intheir size until the process of the pelletization iscompleted (16, 29).

When this technique is used for pelletization, itis important to prevent unnecessary agglomerationof the ingredients that may be the cause of unevensize distribution of the pellets

Melt based agglomeration (16, 30)It is similar to that of liquid based agglomeration

but the only difference is that binding material used isin molten form instead of liquid form. Congealingprocess is involved in which congealing materialhelps in the formation of pellets without formation ofliquid based adhesive bridges between the particles.Depending upon the surface moisture contents, vari-ous sized pellets can be formed during this process.

Freeze pelletization

It is a simple but new technique for the prepa-ration of pellets. In this technique drug carrier isused in molten form. Active ingredient is dispersedin the molten carrier that is added dropwise in thecolumn of immiscible liquid. These droplets canmove in both directions, either upwards or down-wards depending upon the densities of the material.Drug carrier can be hydrophilic or hydrophobic andmelted at the temperature 5 to 10OC greater than itsmelting point. Liquid column is divided into twoparts. One part with temperature range of 25 to100OC and send portion with 0 to ñ40OC, used for thesolidification of melted material. In order to main-tain this temperature cooling mixture of acetone anddry ice is used (25, 31).

Depending upon the nature of the carrier, twotypes of apparatus are available for pelletization thatare Freeze Pelletizer I and Freeze Pelletizer II.

In Freeze Pelletizer I, water soluble materialand low melting point materials are used for exam-ple: polyethylene glycol (PEG), polyvinyl alcohol(PVA), dextrose and maltose etc. are used. Low den-sity liquids are used for column such as mineral oil,vegetable oil and silicon oil. In Freeze Pelletizer II,

hydrophobic carriers are used such as glycerylpalmitostearate, glyceryl behenate and glycerylmonostearate. In column, high density hydrophilicliquids like ethyl alcohol, glycerine, polyethyleneglycol and water are used (29, 32, 33).

CHARACTERIZATION OF PELLETS

Pellets size

Determination of pellets size is important as ithelps to determine the flow properties of the pellets.Particle size also affects the release kinetics of thedrug from the pellets. Various techniques can beused for the particle size determination. The simpleand easy method is the sieving by selecting suitablemesh size shaken with mechanical shaker. Verniercalliper can also be used for size determination (34).

Flow properties

Flow properties can be measured using differ-ent techniques including, angle of repose, Carrísindex, Hausnerís ratio. Flow properties can give aclue of uniform size distribution (34, 35).

Surface morphology

Surface morphology and cross section of theformulated pellets can be observed by using scan-ning electron microscopy. Other techniques includeoptical microscopy and application of non-contract-ing laser profile meter for the examination ofsmoothness of the pellets surface (35).

Surface area

Surface area is considered important featureespecially when coating of the pellets is required.Surface area has direct relation with size and shapeof the pellets. Gas adsorption technique is used forthe determination of specific surface area (36, 37).

Friability

Friability test is performed to evaluate themechanical and tensile strength of the pellets. Tabletfriabilator is used for measurement of friability.Fluidized bed with Wurster insert using stream of aircan be used for friability determination (35, 36).

Pellets shape

Shape of the pellets is another important char-acter of the pellets. Pellets should be of sphericalshape. Different methods can be used for the deter-mination of shape of the pellets. Microscopy andstereomicroscopy can be used. An indirect methodthat is angle of repose can also be used for the indi-cation of spherical shape of the pellets (37, 38).


CONCLUSION

Nowadayís special concern is with the devel-opment of novel drug delivery system and pelletiza-tion is one of them. Pellets with their flexibility informulation design, application and method ofpreparation are suitable dosage form for the deliveryof drug in immediate, intermediate and sustainedrelease form. It is concluded that pelletization is aneffective technique for the achievement of highpatient compliance which has the prime importancein dosage form development.

Acknowledgment

Corresponding author (Muhammad Zaman) isvery thankful to his co-authors for helping him incompletion of this review.

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Received: 30. 10. 2015


Vitamin D ñ metabolism, mechanism of action

Vitamin D, discovered in 1922 by McCollum,was hitherto known mainly for its impact on boneand calcium metabolism. The discovery of vitaminD receptors (VDRs) in most human extraskeletalcells suggested that vitamin D plays a role in othersystems and organs in the body (1).

Vitamin D3 (cholecalciferol) is produced from7-dehydrocholesterol contained in the skin follow-ing exposure to UVB radiation (290-315 nm). It isalso absorbed from dietary sources as vitamin D2(ergocalciferol) or vitamin D3. Vitamin D-bindingprotein (VDBP) facilitates the transport of ergo- andcholecalciferol via the vascular system into the liverwhere both compounds are metabolized. Both com-pounds undergo a two-staged process of activationto 25-hydroxyvitamin D3 (calcidiol, 25(OH)D)using 25-hydroxylases CYP2R1 and CYP27A1

(hepatic enzymes containing cytochrome P450) andfurther to 1α,25-dihydroxyvitamin D3 (calcitriol,1α,25(OH)2D), using CYP27B1, 1α-hydroxylase ñan enzyme present in kidneys, as well as inextrarenal tissues. For the sake of clarity, thedescription of aforementioned transformation doesnot include numerous other reactions that lead tometabolites that are either inactive or less activethan calcitriol, such as C3-epimers (1, 2).

Calcitriol is a biologically active form of vita-min D classified as one of the steroid hormones.Being transported to multiple organs, it interactswith cells through its ability to activate specificVDRs.

The VDR gene is located within the longer armof chromosome 12 (12q13.1) and consists of 14exons amounting to a total of ca. 75kb of genomicDNA (2, 3). The relationship between vitamin D and

VITAMIN D IN ALZHEIMERíS DISEASE ñ PROPHYLAXIS OR THERAPY?

BEATA M. GRUBER-BZURA*

Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Che≥mska 30/34 St., 00-725 Warszawa, Poland

Abstract: The pleiotropism of vitamin D is due to the presence of vitamin D receptor in the cells of nearly alltissues and organs within the human body, including the CNS. Multiple evidence is available to support neu-roprotective properties of vitamin D. These include, for example, the presence of 25(OH)D-1α-hydroxylase, anenzyme responsible for production of calcitriol, within the human brain. Among its other activities, calcitriolmodifies production and release of neurotrophic factors, affects expression of genes associated withGABAergic signaling and stimulates biosynthesis of catecholamines. Antioxidative and anti-inflammatoryproperties were also demonstrated in research studies. By confronting the known pathomechanisms ofAlzheimerís disease (AD) and the mechanism of action of vitamin D, one may propose that systemic insuffi-ciency of vitamin D is a potential risk factor of AD. Studies conducted to date confirm the inverse relationshipbetween serum calcidiol levels and the risk of dementia diseases, including AD. Elevated cerebrospinal fluidlevel of VDBP, a vitamin D binding protein that is also responsible for elimination of β-amyloid peptide (Aβ),a pathogenic factor characteristic for AD, is considered to be a potential marker of AD. Reduction in Aβ lev-els within the CNS is the most important therapeutic target in the treatment of AD. Animal studies confirmedthe impact of vitamin D-enriched diet on the reduction in amyloid deposits, Aβ peptide levels and inflammato-ry reactions as well as on the increase in the level of neurotrophic factor within the brains of Aβ protein pre-cursor (AβPP) ñ transgenic mice. In case of AD, the purposefulness of initiating treatment before the onset ofclinical symptoms is being highlighted. Vitamin D is worth consideration since by inducing the expression ofVDR gene it leads, among others, to the silencing of the transcription of the gene encoding the AβAPP and thusinhibits its cleavage into peptides that form amyloid deposits. Despite the fact that at current state vitamin Dcan hardly be considered a therapeutic agent with an established efficient dose in AD, authors of studies sug-gest that it is important in AD prophylaxis in elderly patients with age-related reduction of serum calcidiol lev-els.

Keywords: vitamin D, neuroprotection, Alzheimerís disease

1427


1428 BEATA M. GRUBER-BZURA

the pathogenesis of numerous diseases, includingneurodegenerative diseases, is based on the poly-morphism within the VRD gene as FokI, BsmI,Tru9I, EcoRV, ApaI, TaqI, and Cdx2 (2).

It was demonstrated that some organs are capa-ble of producing calcitriol by themselves for subse-quent use in proliferation processes or in differenti-ation of regulation of immune response (2). Thediverse systemic functions of vitamin D understoodas interactions of its active metabolite, calcitriol, aredue to the nuclear vitamin D receptors affecting theexpression of genes as well as to induction of certaineffects via a non-genomic pathway using the mem-brane-associated rapid response steroid-bindingreceptor (MARRS) dedicated to binding vitamin Dcontained within caveolae (2, 4).

The pleiotropism of vitamin D is due to thepresence of vitamin D receptor in the cells of nearlyall tissues and organs within the human body,including osteoblasts, thyroid, muscle tissue, stom-ach, skin, adrenal glands, ovaries, retina, lungs,intestines, bone marrow, or brain, with the highestexpression being observed within the hypothalamusand large neurons of substantia nigra (3, 5).

Vitamin D and neuroprotection

Multiple evidence is available to support neu-roprotective properties of vitamin D. These include,for example, the presence of CYP enzymes or25(OH)D-1α-hydroxylase, an enzyme responsiblefor production of calcitriol, within the human brain.Calcitriol modifies production and release of neu-rotrophic factors, as well as synthesis of neuromedi-ators such as acetylcholine. It was also shown toaffect expression of genes associated withGABAergic signaling and stimulate biosynthesis ofcatecholamines (2, 6). Due to the stimulation of theexpression of genes associated with the synthesis ofcalcium-binding proteins and simultaneous reduc-tion in the expression of calcium channels asobserved in rat hypothalamic cell cultures, vitaminD, referred to as the ìforgotten neurosteroidî,reduces the intake of neurotoxic excess of calciumions into the neurons. The results of the studies arealso suggestive of antioxidative properties of cal-citriol, e.g., by increasing the cellular glutathionelevels or inhibiting the synthesis of iNOS (induciblenitric oxide synthase) responsible for the high levelsof neurotoxic nitric oxide under hypoxic conditions.Ability of calcitriol to reduce the levels of nitricoxide as well as TNF-α and interleukin-6 asobserved in EOC13 murine microglial cells deter-mines the direct anti-inflammatory activity of thiscompound (2, 6). All the aforementioned properties

of the active form of vitamin D justify its classifica-tion as a neuroprotective agent.

Vitamin D in Alzheimerís disease

Alzheimerís disease (AD) is one of dementiadiseases and is considered widespread amonghumans over the age of 65. According toChakrabarti et al. (7), the worldwide number ofcases was estimated at 24 million subjects in 2011.This number may be doubled by year 2030.

Brain changes typical for AD consist in forma-tion of Aβ senile plaques and neurofibrillary degen-eration, with modified tau protein playing the cru-cial role. Deposits of Aβ are formed along cerebralblood vessels. Reduction in the number of neuronsand synapses, particularly within the cerebral cortex,is observed (8). There are several hypotheses thatattempt to explain the pathogenesis of AD, includ-ing neurotransmitter efficiency, disturbed energymetabolism, oxidative stress and changes in mitoticsignaling, or amyloid cascade involving the forma-tion of Aβ. Aβ is formed by proteolytic cleavage ofAβAPP by β- and γ-secretases. AβAPP is encodedby APP, PSEN1, and PSEN2 (presenilin 1 and 2)genes. Besides forming neurotoxic aggregates con-sisting of 39-42 amino acids that accumulate insenile plaques, Aβ directly contributes to the forma-tion of peroxides and activates cells convertingnitric oxide into peroxynitrite radicals. AD is alsoassociated with specific isoforms of apolipoproteinE (APOE) ñ a component of chylomicrons and verylow density lipoproteins (VLDL) involved in cho-lesterol transport. One of the isoforms, APOE-ε4,stimulates deposition of Aβ and formation of senileplaques although estimated risk of correlationbetween the presence of allele ε4 and AD differsbetween study populations and depends on the fre-quency of occurrence of the APOE-ε4 isoform (7, 9,10).

As many as 95% cases of AD are sporadic,late-onset cases while early-onset familial disease isresponsible for only 5% of cases. In the first case,the APOE gene is considered to be the marker ofdisease progression. Recent genomic studiesallowed for identification of several other genes thatmight be associated with genetic presentation ofAlzheimerís disease. These include, for example,CR 1, SORL 1, PICALM, and CLU genes (7, 10).

As a result of long-term prospective studies aswell as cross-sectional retrospective studies, severalother extragenomic risk factors of sporadic AD wereidentified, including hypercholesterolemia, type IIdiabetes, hyperhomocysteinemia, elevated levels ofinflammatory cytokines, disturbed circulating

Vitamin D in Alzheimer's desease - prophylaxis or therapy? 1429

adipokine levels and vitamin D deficiency (7). Theearly-onset familial disease is a result of a rare com-bination of mutations within three genes: APP,PSEN1, and PSEN2 (7, 8).

Studies conducted to date confirm the inverserelationship between serum calcidiol levels and therisk of dementia diseases, including AD. The resultsof meta-analysis covering the period between theyear 2010 and February 2015 as presented by Shenand Ji (11), show that the risk of AD is 21% higherin individuals with calcidiol levels of below 50nmol/L as compared to individuals with calcidiollevels of above that value. Littlejohn et al. (12) pre-sented the results of studies conducted over a periodof 5.6 years in years 1992/1993-1999 in a group of1650 elderly patients. Multifactorial adjusted riskfactors of AD calculated on the basis of Cox propor-tional hazards model for participants who wereseverely 25(OH)D deficient (< 25 nmol/L) and defi-cient (≥ 25 nmol/L to < 50 nmol/L) compared to par-ticipants with concentrations ≥ 50 nmol/L were 2.22(CI ≥ 1.02-4.83 at p ≥ 95%) and 1.69 (CI ≥ 1.06-2.69at p ≥ 95%), respectively. Similar results wereobtained in Denmark in a 30-year prospective studyreported by Chakrabarti et al. (7). Annweiler et al.(13) report that administration of more than 800 IUof vitamin D/day over 7 years reduced the risk of ADby a factor of 5. In their retrospective study in 102patients with mild AD, Chaves et al. (14) demon-strated that vitamin D supplementation delayed thedevelopment of acute AD by about a year.

By confronting the known pathomechanismsof AD and the mechanism of action of vitamin D,one may propose that systemic insufficiency of vita-min D is a potential risk factor of AD. Based on thecurrent knowledge, in their study titled ìWhy vita-min D in Alzheimerís disease? The hypothesisî(15), Gezen-Ak et al. defined AD to be a conse-quence of long-term disturbance of hormonal equi-librium with relation to a secosteroid vitamin D.Based on the available studies, elevated CSF levelof VDBP, which is also responsible for eliminationof Aβ, is considered to be a marker of AD (16, 17).Relationships were also demonstrated between theVDR gene polymorphism in alleles TaqI, ApaI,Tru91, BsmI, and FokI, and the incidence of AD;however, in the case of the first two of these alleles,the relationship appears to be population-dependent(3, 7, 18). On the other hand, Wang et al. (19) intheir studies encompassing 492 cases of late-stageAD and 496 control subjects demonstrated a single-nucleotide polymorphism (SNP) within the promot-er region of the VDR gene at the Cdx-2 transcriptionfactor binding site in AD patients (7, 19).

Direct relationships between vitamin D and thepathomechanisms of AD are also confirmed byinteractions between the VDR and Aβ. Besidesenhancing the expression of vitamin D degradationenzyme, Aβ silences the expression of VDR.Dursun et al. (20) observed a protective effect inrelation to cytotoxicity and apoptosis as well asincreased VDR expression as a result of exposing aprimary culture of cortical neurons to vitamin D. Inaddition, the same authors demonstrated that vita-min D stabilizes Aβ-induced iNOS levels via theVDR while omitting the MARRS-dependent path-way (21). The authors suggested that use of this spe-cific interaction in terms of vitamin D supplementa-tion could be made in both treatment and preventionof AD. This suggestion is also supported by theresults of studies conducted by Masoumi et al. (22)who demonstrated that calcitriol strongly stimulatesphagocytosis of Aβ, thus protecting the cells fromamyloid-induced apoptosis.

Interactions between calcitriol and Aβ thatdetermine the importance of vitamin D in both pre-vention and treatment of AD, occur probably alongboth genomic and non-genomic pathways. In theformer case, as demonstrated by Ito et al. (23) usingthe example of rat brain capillary endothelial cells,elimination of Aβ from brain into the blood occurswith a complex formed by VDR and a transactiva-tor, retinoid X receptor (RXR) having the propertiesof a transcription factor. The authors observed thepresence of this complex within the cells. Non-genomic interactions stem from calcitriolís impacton cAMP levels. It was demonstrated that this activeform of vitamin D increases intracellular cAMP lev-els and thus activates the MEK-MAPK-ERK cas-cade. This signaling pathway is involved in elimina-tion of Aβ from the brain into the blood.

The study published by Durk et al. (9) explainsin more detail the mechanism triggered by the VDRtransactivation complex. Using an inhibitor of theATP-dependent membrane pump P-gp, the authorswere able to demonstrate the involvement of thistransporter in the elimination of amyloid from cells.The exposure of transgenic mouse AD models to thecalcitriol dose of 2.5 µg/kg i.p. was associated withincreased expression of both VDR and P-gp.Simultaneously, Aβ levels were reduced, particular-ly within the hypothalamic region. Early and short-term supplementation of mice (every two days, for 8days) with the active form of vitamin D before theformation of amyloid deposits led to reduction inlevels of both the soluble form and the few insolubledeposits of that protein. This is interesting since therole of P-gp consists mainly in elimination of the

1430 BEATA M. GRUBER-BZURA

soluble form of amyloid. At the same time, calcitri-ol supplementation of mice at an advanced stage offorming insoluble Aβ deposits was not effective inreducing the quantity of this deposits and could onlyreduce the soluble form levels. Reduction in Aβ lev-els was reflected in better conditioned fear memoryin the test animals. In addition, the authors demon-strated that in animals fed with vitamin D-deficientdiet, cerebral expression of P-gp was reduced, sug-gesting an indirect mechanism of limitation of cog-nitive function observed in older animals.

In the light of the above studies on the supple-mentation regimen, the authors came to a conclusionthat the replenishment of vitamin D levels is moreimportant in AD prevention before Aβ being toaccumulate, particularly in elderly patients in whomthe level of the active form of this vitamin is reducedwith age.

The results of the studies of the effect of vita-min D on the development and progression of ADare also indicative of other mechanisms underlyingthe inhibition of the disorder. These include VDR-mediated stimulation of the release of neurotrophicfactors within hypothalamic neurons as well as theeffect of calcitriol or its combination with ω-3 acidson macrophagesí ability to carry out phagocytosis ofamyloid deposits, inhibit Aβ-induced apoptosis inmononuclear cells, or silence the transcriptions ofgenes encoding pro-inflammatory cytokines asdemonstrated in blood cells collected from supple-mented and non-supplemented AD patients. Theseeffects were also correlated with improved cognitiveabilities in patients (24).

Animal studies confirmed the impact of vita-min D-enriched diet on the reduction in Aβ levelsand inflammatory reactions as well as on theincrease in the level of neurotrophic factor withinthe brains of (AβPP)-transgenic mice.

Reduction in Aβ levels within the CNS is themost important therapeutic target in the treatment ofAD. In the light of these properties, calcitriolbecame an important candidate for a therapeutic aswell as prophylactic agent.

Drugs that have been used for many years inAD patients, such as memantin or cholinesteraseinhibitors, are symptomatic medications that delaythe progress of the disease rather than treat it and arededicated only to patients with developed mild andacute disease, whereas it has been pointed out that itis purposeful to initiate the treatment before theonset of clinical symptoms (13). Vitamin D is worthconsideration since by inducing the expression ofVDR gene it leads, among others, to the silencing of

the transcription of the gene encoding AβPP andthus inhibits its cleavage into peptides that formamyloid deposits.

Combination treatment using vitamin D is con-sidered for patients with advanced AD. In vitro stud-ies demonstrated that in combination with meman-tin, vitamin D reduced the degree of degeneration ofthe axons of cortical neurones exposed to Aβ or glu-tamate as compared to the response observed in cul-tures exposed to either of these compounds sepa-rately (26).

Pharmacokinetic characteristics of calcitriolmay be a guidance for practical use of calcitriol inAD treatment. Maximum blood concentration ofcalcitriol is achieved within 2 h after intraperitonealadministration and subsequently drops to the base-line level within 24 h. This pharmacokinetic profilewas confirmed in the studies conducted by Ito et al.(23) who observed the minimum levels of Aβ in thebrains of mice 24 h after intraperitoneal administra-tion of 1 µg of calcitriol. No reduction was observedafter 48 or 72 h. Therefore, in order to achieve thedesired reduction in cerebral amyloid levels, multi-ple doses of calcitriol should be used. This, in turn,is associated with a risk of adverse effects, namelyhypercalcemia, which was observed in mice fol-lowing daily administration of more than 0.1 µg for2 weeks. Unfortunately, according to unpublishedresults obtained by the authors, vitamin D analogssuch as paricalcitol, although leading to a lowerlevel of hypercalcemia as compared to calcitriol,are also less effective in the reduction of amyloidlevels.

Summary

As shown, vitamin D has been studied in thecontext of neurodegenerative diseases for manyyears. Its activity at the molecular levels involvesmany pathways common with those involved in thepathomechanism of AD and thus fully justifiesresearch on the use of vitamin D in the preventionand/or treatment of the disease. Despite the fact thatat current state vitamin D can hardly be considereda therapeutic agent with an established efficient dosein AD, authors of studies suggest that it is importantin AD prophylaxis in elderly patients with age-relat-ed reduction of serum calcidiol levels.

At the same time, the growing investigativecapabilities and increasingly detailed research topicsallow for more precise answers to the questionappropriately formulated by Gezen-Ak et al. (15):ìwhy should we seriously consider ìsimpleî vitaminD as a ìfundamental factorî in AD?î.

Vitamin D in Alzheimer's desease - prophylaxis or therapy? 1431

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7. Chakrabarti S., Khemka V.K., Banerjee A.,Chatterjee G., Ganguly A., Biswas A.: AgingDis. 6, 282 (2015).

8. Gruber B.M.: Akt. Neurol. 9, 52 (2009).9. Durk M.R., Han K., Chow E.C.Y., Ahrens R.,

Henderson J.T. et al.: J. Neurosci. 32, 7091(2014).

10. Gruber B.M.: Postepy Hig. Med. Dosw. 65, 542(2011).

11. Shen L., Hong-Fang J.: Nutr. J. 14, 76 (2015).12. Littlejohn T.J., Henley W., Lang I.A.,

Annweiler C., Beaucher O. et al.: Neurology 83,920 (2014).

13. Annweiler C., Karras S.N., Anagnostis P.,Beauchet O.: Front. Pharmacol. 5, 6 (2014).

14. Chaves M., Toral A., Bisonni A., Rojas J.I.,Fern·ndez C. et al.: Vertex 25, 85 (2014).

15. Gezen-Ak D., Yilmazer S., Dursun E.: J.Alzheimers Dis. 40, 257 (2014).

16. Bishnoi R.J., Palmer R.F., Royall D.R.: J.Alzheimers Dis. 43, 37 (2015).

17. Zhang J., Sokal I., Peskind E.R., Quinn J.F.,Jankovic J. et al.: Am. J. Clin. Pathol. 129, 526(2008).

18. Lee Y.H., Kim J.H., Song G.G.: Neurol. Sci. 35,1947 (2014).

19. Wang L., Hara K., Van Baaren J.M., Price J.C.,Beecham G.W. et al.: Neurobiol. Aging 33,1844.e1-9 (2012).

20. Dursun E., Gezen-Ak D., Yilmazer S.: J.Alzheimers Dis. 23, 207 (2011).

21. Dursun E., Gezen-Ak D., Yilmazer S.: J.Alzheimers Dis. 36, 459 (2013).

22. Masoumi A., Goldenson B., Ghirmai S.,Avagyan H., Zaghi J. et al.: J. Alzheimers Dis.17, 703 (2009).

23. Ito S., Ohtsuki S., Nezu Y., Koitabashi Y.,Murata S., Terasaki T.: Fluids Barriers CNS 8,20 (2011).

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Received: 10. 12. 2015


Depression is a devastating, widespread condi-tion affecting 15ñ20% of the population. It maycause significant functional impairment. In spite ofvariety of available antidepressant drugs, more than30% of the patients with major depressive disorder(MDD) still cannot achieve neither adequateresponse nor remission (1).

Both DSM-5 and ICD-10 diagnostic criteriaunderline the significance of symptoms resultingfrom cognitive impairment. DSM-5 specify: dimin-ished ability to think or concentrate or more indeci-siveness present nearly every day (indicated byeither subjective report or observation made by oth-ers), psychomotor agitation or retardation (non-sub-jective, reported by others) (2). In turn, ICD-10 ñindicate poor concentration, substantial psychomo-

tor retardation or agitation (3). Nonetheless, the roleof cognitive dysfunctions seem to remain underesti-mated in case of depressive disorders, thus they areperceived as therapeutic target (4).

Vortioxetine is an antidepressant drug suitablefor therapy in various types of depression: severe,anxiety-associated, of elders. It acts equally strongas SNRIs or agomelatine and has favorable effectson cognitive functioning (4, 5).

Vortioxetine is a relatively new, multi-func-tional agent. Regarding this, it resembles nefa-zodone, trazadone, vilazodone and tedatioxetine.The drug code name is Lu AA21004 (6, 7). Thecompound has been developed by Lundbeck andTakeda alliance and its approval for the treatment ofmajor depressive disorder in adults was announced

VORTIOXETINE ñ THE NEW ANTIDEPRESSANT AGENT WITH PROCOGNITIVE PROPERTIES

EWELINA DZIWOTA* and MARCIN OLAJOSSY

2nd Department of Psychiatry and Psychiatric Rehabilitation, Medical University of Lublin, G≥uska 1, 20-439 Lublin, Poland

Abstract: Depression symptoms resulting from cognitive function impairment are emphasized by both DSM-5 and ICD-10 diagnostic criteria for major depressive disorder and depressive episodes. Nonetheless, the roleof cognitive dysfunctions seem to remain underestimated in case of depressive disorders, thus they are rarelyperceived as therapeutic target. Vortioxetine is a relatively new, multi-functional agent. With its unique prop-erties and strong affinity towards serotonin transporter (5-HTT), vortioxetine is a modulator and stimulator ofserotonergic transmission. Vortioxetine is an antidepressant drug suitable for therapy in various types of depres-sion: severe, anxiety-associated, and of elders. It acts equally strong as SNRIs or agomelatine and has favorableeffects on cognitive functioning. Although vortioxetine has not undergone comprehensive preclinical testing,the available data indicate that this particular agent may be more advantageous in terms of its procognitiveeffects, as compared to other drugs ñ which often seemed to be analogous in preclinical and clinical testing. Invitro examination of hippocampal pyramidal cells revealed that vortioxetine improves both synaptic transmis-sion and neuroplasticity responsible for memory and learning patterns. Contrary to fluoxetine, the long-termtreatment with use of vortioxetine on mice resulted in enhanced visual and spatial memory, along with reducedoccurrence of typical depressive behavior. In addition, vortioxetine is a very first drug efficiently augmentingcognitive function in adults diagnosed with severe depressive episode, irrespective of its curative potential onthe affective sphere. It may exert even stronger direct effect (assessed with DSST) on cognitive functions thanduloxetine. With its supplementary capacity of acting directly on several subtypes of serotonin receptors, vor-tioxetine is certainly more than just a SSRI. It has been proved that it is as effective as venlafaxine and moreefficient than agomelatine in MDD treatment, additionally exerting procognitive effects. In addition, vortioxe-tine may be beneficial in overcoming sexual dysfunction in patients, who have been suffering from such con-dition as a result of treatment with other antidepressant agents. The drug is generally well tolerated with themost prevalent side effects being mild to moderate nausea along with (mostly transient) headaches.Vortioxetine may significantly improve the quality of life in patients suffering from depression.

Keywords: vortioxetine, depression, cognitive dysfunction

1433


1434 EWELINA DZIWOTA and MARCIN OLAJOSSY

in September 2013 by FDA, and in October 2013 byEMA (European Medicines Agency) (7, 8).

Pharmacodynamic and pharmacokinetic proper-

ties of vortioxetine

Vortioxetine is, in terms of chemistry, a deriv-ative of arylpiperazine: 1-[2-(2,4-dimethyl-phenyl-sulfanyl)-phenyl]-piperazine (6). The molecularweight and molecular formula of vortioxetine are:298.45 g/mol and C18H22N2S, respectively. Thebioavailability after oral administration amounts to75% and the mean half-life equals 57 h (6, 7). Thepeak plasma concentration of 9-33 ng/mL is reachedafter 7-11 h, under assumption of administering 5-10 mg daily doses (9). Vortioxetine is metabolizedby several liver P450 cytochromes: CYP2D6,CYP3A4/5, CYP2C19, CYP2C9, CYP2A6,CYP2C8 and CYP2B6 in particular (9, 10).

With its unique properties and having strongaffinity towards serotonin transporter (5-HTT), vor-tioxetine is a modulator and stimulator of serotoner-gic transmission. Additionally, it inhibits ñ Ki = 1.6nM, serotonin transporter (SERT).

Vortioxetine presents agonistic effect towards5-HT1A receptor (Ki = 15 nM), with high levels ofintrinsic activity (IA = 80%) (6, 11). The compoundis a partial agonist of 5-HT1B receptor (Ki = 33 nM)(9, 11) and the antagonist of 5-HT1D, 5-HT3 and 5-HT7 receptors (Ki = 19 nM) (8, 11). Distribution ofthe 5-HT7 receptors in central nervous system indi-cates for their regulatory role in cognitive function-ing, attitude, perception of pain, circadian rhythmand sleep (12). Serotonin is an evolutionary trans-mitter, which modulates numerous functions of thecentral nervous system, e.g.: mood, anxiety, stress,aggression, feeding, cognition or sexual behavior(13).

Previously described effect of vortioxetine onserotonin transmission may underlie the concept ofexerting impact on neural pathways controlled byother modulators, responsible for attitude and cogni-tive capacity, including norepinephrine (NE),acetylcholine (ACh) and glutamate. In preclinicalstudies, vortioxetine increased the extracellular lev-els of 5-hydroxytryptamine, NE, ACh, HA anddopamine in cerebral regions involved in both moodmodulation and cognitive functioning (9).

As far as potential side effects are concerned,the vortioxetineís affinity towards β1 adrenergicreceptor (Ki = 46 nM) should be considered (6).

Clinical application of vortioxetine

Compared to placebo, vortioxetine proved tobe more effective in treatment of the Major

Depressive Disorder rated with use of HDRS (TheHamiltonian Depression Rating Scale) or MADRS(The Montgomery Asberg Depression RatingScale), as was visible while comparing scoresachieved at the beginning, after response to therapyand at the remission, respectively (14, 15).

Alvarez et al. determined the antidepressantefficacy of vortioxetine in contrast to venlafaxineand placebo, in a randomized, controlled, double-blind clinical trial. Initially, all the patients in thestudy presented with min. 30 MADRS pointsdepression. Drug tolerance for 5 mg dose of vor-tioxetine and placebo were comparable, and 10 mgdose was even better tolerated than the 225 mg doseof venlafaxine (6).

The effectiveness of vortioxetine in depressiontreatment was further evaluated by Baldwin et al.(16). The 8-week-long multicentre randomized clin-ical trial was designed to confront 2.5, 5 and 10 mgvortioxetine doses with placebo and 60 mg-dose ofduloxetine. The mixed model repeated measuresanalysis proved higher efficacy of 5 and 10 mg vor-tioxetine and 60 mg duloxetine, as compared toplacebo (6, 15, 16).

Furthermore, Henigsberg et al. compared anti-depressant potential of 1, 5 and 10 mg doses of vor-tioxetine with placebo. Ten mg dose of vortioxetineproved to be an effective treatment of depression interms of primary endpoint of the study ñ improve-ment in the Hamiltonian scale. A global assessmentwith use of HDRS, clinical global impression ñCGI-I and MADRS demonstrated higher efficacy ofvortioxetine over placebo, at all of the examineddoses (9, 17). In addition, Boulenger et al. showedthe efficiency of vortioxetine in prevention of recur-rent episodes of depression (18).

Furthermore, vortioxetine has positive effectson anxiety reduction (5). A trial conducted inEurope and Africa by Bidzan et al. aimed to assessthe curative potential of vortioxetine in generalizedanxiety disorder (GAD). Three hundred and onepatients diagnosed with GAD were included in thestudy, all of them having > 20 points in HAM-Ascale. The MMRM analysis indicated a significantadvantage of vortioxetine over placebo (19).

Analogous studies were carried by Rothschildet al. in USA. Patients were randomized into 2groups, receiving: 5 mg of vortioxetine or placebo.Anxiety levels were measured with use of HAM-Ascale. After 8 weeks of treatment regimen, no dif-ferences were observed between tested drug andplacebo with reference to primary endpoints of thestudy. Such discrepancies between the resultsobtained by Rothschild and Bidzan may resulted

Vortioxetine - the new antidepressant agent with procognitive properties 1435

from: higher mean anxiety level and greater percentof previously treated patients in Bidzanís study,smaller percent of untimely patient resignations inthe same study, and the majority of patients enrolledin Bidzanís trial being Caucasian ñ thus, accordingto STAR*D study, having greater probability ofachieving remission. In turn, Baldwin et al. evaluat-ed the role of vortioxetine in prevention of recurrentGAD. Introducing vortioxetine was linked to signif-icantly lower risk of recurrent episodes (6, 20).

Vortioxetine procognitive potential in preclinical

Although vortioxetine has not undergone com-prehensive preclinical testing, the available dataindicate that this particular agent may be moreadvantageous in terms of its procognitive effects, ascompared to other drugs ñ which often seemed to beanalogous in preclinical and clinical testing (21).Procognitive properties of vortioxetine have beendepicted by various preclinical trials. In vitro exam-ination of hippocampal pyramidal cells revealed thatvortioxetine improves/increases both synaptic trans-mission and neuroplasticity responsible for memoryand learning patterns, whereas escitalopram present-ed no such procognitive potential. Using the cogni-tive-behavioral models (new object recognitiontests), vortioxetine caused memory improvement inrats. Additionally, it increased the levels of acetyl-choline and histamine in medial prefrontal cortex.This finding is of particular importance consideringcognitive capacity, as acetylcholine is responsiblefor memory and learning, while histamine plays rolein attention, alertness and memory (9, 22).

Westrich et al. dedicated their trial to assessvortioxetineís modulatory potential on various 5-HT7 receptors in terms of its influence on circadianrhythm and memory in rats. Object RecognitionTest was among exploited methods. The studyrevealed that vortioxetine exerts substantial effectson circadian rhythm and episodic memory correc-tion in rats, mainly due to its antagonistic propertiestowards 5-HT7 receptors (23).

Yan Li et al. compared the effect of fluoxetineand vortioxetine action on cognitive functioning,affective behavior, cerebral stem cell proliferation,growth factor levels and gene expression in middle-aged mice. The visual-spatial skills are age-relatednot only in human, but also in animal models. Thestudy indicated the existence of age-related visual-spatial memory dysfunction in healthy middle agedmice. Contrary to fluoxetine, the long-term treat-ment with vortioxetine resulted in enhanced visual-spatial memory skills, along with reduced occur-rence of typical depressive behavior in mice (24).

Procognitive effects presented in clinical trials

Vortioxetine is a very first drug that efficientlyaugments cognitive function in adults diagnosedwith severe depressive episode, irrespective of itscurative potential on the affective sphere (9).

In a randomized, placebo-controlled trial on 24healthy volunteers, neither short nor long-term treat-ment with 10 mg of vortioxetine hindered psy-chomotor or cognitive functions. At the same time,even single administration of 30 mg of mirtazapinedistorted such abilities. These results clearly provethat vortioxetine has no negative effect on cognitivefunctions (9).

Several clinical trials were dedicated to verifythe independent procognitive properties of vortioxe-tine. One of them was a randomized, placebo-con-trolled, double-blind study concerning adult patientsdiagnosed with depression and MDE and MDRSscore ≥ 26. They received placebo, or either 10 or 20mg of vortioxetine over 8-week period. The primaryendpoint comprised change in combined score fromtwo neuropsychological tests: DSST (the DigitSymbol Substitution Test ñ which assessed the rate ofprocessing information, executive function and alert-ness) and RAVLT (the Rey Auditory VerbalLearning Test ñ assessing memory and learningcapacity), achieved after 8 weeks of treatment ñ ascompared to initial state. This particular study includ-ed 193, 204 and 194 patients receiving 10 or 20 mg ofvortioxetine or placebo, respectively. As compared toplacebo, both prescribed doses of vortioxetine pre-sented statistically significant outcome in comparisonwith the initial point. Further analysis based on indi-vidual neuropsychological tests revealed the advan-tage of both doses of vortioxetine over placebo (6, 9).

A randomized, placebo-controlled study onvortioxetine and duloxetine had been conducted byMahableshwarkar et al. It assessed vortioxetineíspotential effect on cognitive functioning in patientswith depression, who reported deterioration of suchskills. The trial outcome indicated the advantage ofvortioxetine in 10-20 mg daily doses over placebo,with reference to initial DSST, PDQ and CGI-Iscores. The results of University of San DiegoPerformance-Based Skills Assessment as wellstressed the substantial improvement of functions inpatients treated with vortioxetine. The most oftencompared agent ñ duloxetine, exhibited no signifi-cant difference from placebo in terms of DSST andSan Diego Performance-Based Skills Assessment.However, it considerably improved in PDQ andCGI-I (9, 20).

Theuniessen et al. focused on determining theeffect of vortioxetine on cognitive and psychomotor

1436 EWELINA DZIWOTA and MARCIN OLAJOSSY

abilities. The study revealed no unfavorableresponse in terms of driving as well as cognitive andpsychomotor capacity, after either short or long-term treatment with vortioxetine (25).

Katona et al. assessed the antidepressant poten-tial and safety of administering vortioxetine in popula-tion of elder patients. In a 8-week, randomized clinicaltrial, the drug had been compared to placebo and 60mg of duloxetine in patients (mean age of 70.6 years)with recurrent depressive episodes. Both agentsshowed to be more effective than placebo, and vor-tioxetine was better tolerated than duloxetine.Vortioxetine also proved to be more efficient thanplacebo in terms of improving memory, informationprocessing and verbal learning (26). Katona et al. eval-uated the effects of vortioxetine on cognitive functionsin elderly patients diagnosed with MDD. Vortioxetinesignificantly outranked the placebo, resulting in sub-stantial improvement of DSST and RAVLT. In com-parison, duloxetine improved RAVLT, but not DSST.This indicates that vortioxetine may exert greaterdirect effect on cognitive function enhancement(measured with DSST) than duloxetine (26).

While analyzing cognitive dysfunction indepression, current classification should be recalled,which distinguished cold (independent of emotion)and hot (emotion-dependent) cognitive impair-ments,. It is commonly believed that cold cognitivedeficits do not retreat in remission state, and in per-sistent form they may cause poor response to treat-ment with antidepressants (4).

Safety of medication administration

The side-effect profile of vortioxetine is simi-lar to other SSRIs. It comprises nausea, vomiting,diarrhea, headache and vertigos (6). The most preva-lent adverse events were linked to gastrointestinalsystem. Nausea was the most often reported sideeffect, and it seemed to be dose dependent. Nearly32% of patients receiving 20 mg dose of vortioxe-tine suffered from nausea (9). Vortioxetine neitherhad effect on weight gain, nor caused ECG devia-tions (6). Furthermore, 2-week randomized studyconfronting placebo and moxifloxacin with 10 and40 mg of vortioxetine revealed no significantincrease in the QTc (9). Sexual dysfunctionsoccurred less often than in case of venlafaxine (6).The number of spontaneously reported sexual dys-functions from patients who were on vortioxetinetherapy was low both in randomized and open stud-ies. Vortioxetine was compared to escitalopram (10-20 mg doses) in an 8-week, randomized, double-blind study including 447 MDD patients, who hadbeen previously treated with SSRIs causing sexual

dysfunction. Vortioxetine showed a substantialimprovement in CSFQ-14 (Changes in SexualFunctioning Questionnaire Short-Form) in compari-son with the initial results (9, 27).

Exerting additional impact on certain serotoninreceptors, vortioxetine is certainly more than just aSSRI. It acts equally strong as SNRIs or agomela-tine and has favorable effects on cognitive function-ing. Furthermore, it may reduce sexual dysfunctionin patients who reported such problems during treat-ment with other antidepressants. The medication isgenerally well tolerated, with the most prevalentside effect being mild to moderate nausea along withmostly transient headaches (28).

Vortioxetine may significantly improve thequality of life in patients suffering from depression.Florea et al. explored its effect on the quality of lifein such patients. Health Related Quality of Life(HRQoL) had been assessed in adult patients withMDD during 6-8-week long randomized studies ofvortioxetine versus placebo, with use of: 36-pointShort-Form Health Survey, the Quality of LifeEnjoyment and Satisfaction Questionnaire short-form, the EuroQol 5-Dimension Questionnaire and12-point Health Status Questionnaire (in one trial).Patients had been administered 5, 10, 15 or 20 mgdoses. Vortioxetine therapy lead to significantimprovement in HRQoL in these patients (29).

It is possible that this up-and-coming medica-tion will change the future of psychiatric patients bysubstantially improving their quality of life and cog-nitive functioning.

REFERENCES

1. Wang S.M., Han C., Pae C.U.: Expert Opin.Investig. Drugs 24, 445 (2015).

2. Diagnostic and Statistical Manual of MentalDisorders (DSM-5). 5th edn., AmericanPsychiatric Association 2013.

3. The ICD-10 classification of mental and behav-ioural disorders: clinical descriptions and diag-nostic guidelines. World Health Organization,Geneva 1992.

4. Jarema M., Dudek D., Czernikiewicz A.:Psychiatr. Pol. 48, 1105 (2014).

5. Pae C.U., Wang S.M., Han C., Lee S.J., PatkarA.A. et al.: J. Psychiat. Res. 64, 88 (2015).

6. WysokiÒski A., K≥oszewska I.: PostÍpy Psy-chiatrii i Neurologii 22, 187 (2013).

7. Kelliny M., Croarkin P.E., Moore K.M., BoboW.V.: Ther. Clin. Risk Manag. 11, 1193 (2015).

8. Sanchez C., Asin K.E., Artigas F.: Pharmacol.Therapeut. 145, 43 (2015).

Vortioxetine - the new antidepressant agent with procognitive properties 1437

9. Al-Sukhni M., Maruschak N.A., McIntyre R.S.:Expert Opin. Drug Saf. 14, 1291 (2015).

10. Gu E.M., Huang C., Liang B., Yuan L., Lan, T.et al.: J. Chromatogr. B 997, 70 (2015).

11. Stahl S.M.: CNS Spectr. 20, 455 (2015).12. BieÒkowski P., Dudek D., Samochowiec J.:

Psychiatr. Pol. 49, 243 (2015).13. Olivier B.: Eur. J. Pharmacol. 753, 2 (2015).14. Pae C.U., Wang S.M., Han C., Lee S.J., Patkar

A.A. et al.: J. Psychiatry Neurosci. 40, 174(2015).

15. Meeker A.S., Herink M.C., Haxby D.G.,Hartung D.M.: Syst. Rev. 4, 21 (2015).

16. Baldwin D.S., Loft H., Dragheim M.: Eur.Neuropsychopharmacol. 22, 482 (2012).

17. Henigsberg N., Mahableshwarkar A.R.,Jacobsen P., Chen Y., Thase M.E.: J. Clin.Psychiatry 73, 953 (2012)

18. Boulenger J.P., Loft H., Florea I.: J.Psychopharmacol. 26, 1408 (2012).

19. Bidzan L., Mahableshwarkar A.R., Jacobsen P.,Yan M., Sheehan D.V.: Eur. Neuropsycho-pharmacol. 22, 847 (2012).

20. Rothschild A.J., Mahableshwarkar A.R.,Jacobsen P., Yan M., Sheehan D.V.: Eur.Neuropsychopharmacol. 22, 858 (2012).

21. Pehrson A.L., Leiser S.C., Gulinello M., DaleE., Li Y. et al.: Eur. J. Pharmacol. 753, 19(2015).

22. BÈtry C., EtiÈvant A., Pehrson A., S·nchez C.,Haddjeri N.: Prog. Neuro-Psychopharmacol.Biol. Psychiatry 58, 38 (2015).

23. Westrich L., Haddjeri N., Dkhissi-Benyahya O.,S·nchez C.: Neuropharmacology 89, 382(2015).

24. Li Y., Abdourahman A., Tamm J.A., PehrsonA.L., S·nchez C. et al.: Pharmacol. Biochem.Behav. 135, 70 (2015).

25. Theunissen E. L., Street D., H�jer A.M.,Vermeeren A., Oers V.A. et al.: Clin.Pharmacol. Ther. 93, 493 (2013).

26. Katona C., Hansen T., Olsen C.K.: Int. Clin.Psychopharmacol. 27, 215 (2012).

27. Jacobsen P.L., Mahableshwarkar A.R., ChenY., Chrones L., Clayton A.H.: J. Sex. Med. 12,2036 (2012).

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Rive B. et al.: Clin. Ther. 37, 2309 (2015).

Received: 19. 01. 2016


Electron paramagnetic resonance (EPR) spec-troscopy was used by us to examine free radical for-mation in different drugs during thermal sterilization(1-5). Dependence of free radical concentrations incefaclor (1), clarithromycin (1), famotidine (2), bis-muth subgallate (3), isosorbide dinitrate (4), andneomycin (5), on temperature and time of heatingwas observed. In this work, free radicals formed inrosuvastatin thermally sterilized according to PolishPharmacopoeia X (6) and the other norms in phar-macy (7) were tested. Exactly the same temperatureand times of thermal sterilization, recommended bythe pharmaceutical norms, were used for rosuvas-tatin. Microbiological tests of sterilization productsare usually done in practice. In this work, the addi-tional examination with application of electron para-magnetic resonance spectroscopy is proposed.Sterilized drugs should not contain high amount offree radicals, because of their reactivity. Rosu-vastatin is a drug that is an hypolipidemic, belong-ing to the statins (8). Rosuvastatin inhibits competi-tively and reversibly the activity of HMG-CoAreductase. Treatment with rosuvastatin reduced cho-

lesterol synthesis in the liver, which increases thenumber of LDL receptors on hepatocytes. For thisreason there is a greater uptake of cholesterol fromthe plasma and a decrease in LDL and total choles-terol in the blood plasma (8). The best parameters ofthermal sterilization of rosuvastatin were searched,as those with the lowest free radical concentration.We hope that EPR examination of the sterilizeddrugs will be used in pharmaceutical practice. In thiswork, the usefulness of free radical analysis forexemplary drug is presented.

EXPERIMENTAL

Samples

Rosuvastatin (3R,5S,6E)-7-[4-(4-fluorophen-yl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid)(C22H28FN3O6S) was examined in this work.Rosuvastatin was purchased from Sigma-Aldrich,CAS Number 147098-20-2, SML 1264, assay 98%,form - white powder. Optical activity [α]D +12O to+18O (c = 1 cm in methanol : water 1 : 1, v/v).

ANALYSIS

FREE RADICAL FORMATION IN ROSUVASTATIN DURING THERMALSTERILIZATION AT DIFFERENT TEMPERATURES

PAWE£ RAMOS*, SYLWIA JARCO, PIOTR PEPLI—SKI and BARBARA PILAWA

School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Department of Biophysics,

Jednoúci 8, 41-200 Sosnowiec, Poland

Abstract: Rosuvastatin was thermally sterilized at temperatures 160OC during 120 min, 170OC during 60 minand 180OC during 30 min, according to Polish pharmacopoeia norms. Free radicals formed in the heated rosu-vastatin samples were examined by electron paramagnetic resonance (EPR) spectroscopy. The best conditionsof thermal sterilization of rosuvastatin were searched. EPR spectra were measured for the analyzed drug inde-pendent on heating temperature. Decrease of free radical concentrations in rosuvastatin with increasing of heat-ing temperature was stated. The optimal conditions for thermal sterilization of rosuvastatin were temperature180OC and heating time of 30 min and resulted in the lowest free radical concentration. The changes of ampli-tudes (A) and linewidths (∆Bpp) of EPR spectra of the heated rosuvastatin were characteristic for homogeneousbroadening EPR lines. Slow spin-lattice relaxation processes existed in the all rosuvastatin samples. The con-firmation of usefulness of electron paramagnetic resonance spectroscopy in pharmacy was presented.

Keywords: rosuvasatin, free radicals, thermal sterilization, EPR spectra, EPR spectroscopy

1439


1440 PAWE£ RAMOS et al.

Chemical structure of rosuvastatin is shown inFigure 1 (9).

Thermal sterilization

Rosuvastatin was thermally sterilized in the hotair oven with air circulation, the professional steril-izer (Memmert GmbH, Germany). The conditions ofthermal sterilization were in agreement with theserecommended by Polish Pharmacopoeia X (6) andthe norms (7). Rosuvastatin samples were heated attemperatures (T) and times (t): 160OC (T) and 120min (t), 170O (T) and 60 min (t) and 180OC (T) and30 min (t). According to Polish Pharmacopoeia X(6) microorganisms are removed from drugs underthese conditions.

EPR measurements

Electron paramagnetic resonance (EPR)measurements were performed at the X-Band (9.3GHz) with magnetic modulation of 100 kHz. EPR

spectra as the first-derivatives were measured bythe use the EPR spectrometer produced byRadiopan (PoznaÒ, Poland) and the system ofnumerical acquisition - the Rapid Scan Unit ofJagmar (KrakÛw, Poland). Microwave frequencywas measured by MCM101 recorder of EPRAD(PoznaÒ, Poland). The total microwave power pro-duced by klystron was 70 mW. Attenuation waschanged from 15 dB to 0 dB, and the microwavepowers were in the range of 2.2-70 mW. The meas-urements for rosuvastatin were performed for thethermal sterilization products at room temperature5 days after heating. During the measurements, thesamples were located at thin walled glass tubeswith the external diameter of 3 mm. Mass samplesin the tubes were determined by weighing(Sartorius).

EPR spectra analysis

The following parameters of the EPR spectrawere determined: amplitudes (A) [+0.01 a.u.], inte-gral intensities (I) [±0.02 a.u.], linewidths (∆Bpp)[±0.02 mT], and resonance magnetic induction (Br)[±0.01 mT]. The influence of microwave power onamplitudes (A) and linewidths (∆Bpp), wereobtained. Integral intensity was obtained by doubleintegration of the first-derivative EPR spectra andthey were used to determine free radical concentra-tions (N) in rosuvastatin. These parameters areshown in Figure 2.

The resonance magnetic induction (Br) andmicrowave frequency (ν), were used to determine g-factor. g-Factor, which depends on localization ofunpaired electrons in the sample, was calculatedfrom the paramagnetic resonance condition as: g =hν/µBBr, where: h ñ Planck constant, ν ñ microwaveFigure 1. Chemical structure of rosuvastatin (9)

Figure 2. The parameters of the EPR spectra: A ñ amplitude, ∆Bpp ñ linewidth, Br ñ resonance magnetic induction

Free radical formation in rosuvastatin during thermal sterilization at... 1441

frequency, µB ñ Bohr magneton, Br ñ induction ofresonance magnetic field.

The EPR spectra were measured and analyzedby professional spectroscopic programs of Jagmar(KrakÛw, Poland) and LabView (NationalInstruments, USA).

Determination of free radical concentration

Free radical concentrations (N) in thermallysterilized rosuvastatin samples were determined

according to the formula (10-12): N = Nu[(WuAu)/Iu]◊ [I/(WAm)], where: Nu - the number of paramag-netic centers in the ultramarine reference; W, Wu -the receiver gains for the rosuvastatin sample andthe ultramarine; A, Au - the amplitudes of ruby sig-nal for the rosuvastatin sample and the ultramarine;I, Iu - the integral intensities for the rosuvastatinsample and ultramarine, m - the mass of the sample.The reference for free radical concentration wasultramarine with the stable paramagnetic centers. A

Figure 3. EPR spectra of rosuvastatin thermally sterilized at temperatures (a) 160OC (120 min), (b) 170OC (60 min), and (c) 180OC (30 min).B ñ magnetic induction. The measurements were done with microwave power of 2.2 mW


ruby crystal (Al2O3: Cr3) permanently placed in theresonance cavity was used as the second reference.

RESULTS AND DISCUSSION

Free radicals did not occur in the nonheatedrosuvastatin, which did not have unpaired electronsin chemical structure (Fig. 1) (9). Thermal steriliza-tion produced free radicals in rosuvastatin, inde-pendent on the heating parameters. Rosuvastatinthermally sterilized at temperatures: 160OC (120min), 170OC (60 min) and 180OC (30 min) revealedEPR spectra, which are presented in Figure 3 a-c,respectively.

The parameters of the EPR spectra were com-pared in Figure 4 a-b. Amplitudes (A) of the EPR

lines of rosuvastatin heated at temperatures: 160OCduring 120 min, 170OC during 60 min and 180OCduring 30 min are shown in Figure 4a. The highestamplitude (A) characterized the drug heated at tem-perature 160OC (120 min), and the lowest amplitude(A) was observed for EPR spectrum of rosuvastatinheated at temperature 180OC (30 min). All the EPRspectra were broad lines with high values oflinewidths (∆Bpp) in the range 0.51-0.55 mT (Fig.4b). Dipolar interactions between free radicals (10-12) in heated rosuvastatin were responsible for thesehigh values of linewidths.

Free radical concentrations (N) were ~1018

spin/g and depended on temperature of thermal ster-ilization of rosuvastatin. The values of concentra-tions of free radicals (N) in rosuvastatin heated at

Figure 4. The parameters of the EPR spectra of thermally sterilized rosuvastatin: (a) amplitudes (A) and (b) linewidths (∆Bpp).Temperatures (T) and times (t) of thermal sterilization were: T = 160OC and t = 120 min, T = 170OC and t = 60 min and T = 180OC and t= 30 min. Data for the EPR spectra recorded with the low microwave power of 2.2 mW


temperatures: 160OC (120 min), 170OC (60 min) and180OC (30 min) are compared in Figure 5. Thedecrease of free radical concentration (N) in rosu-vastatin with increasing of temperature of thermalsterilization was observed. This correlation may beprobably explained by interactions of the sampleswith oxygen. The results in Figure 5 indicate that thebest conditions of thermal sterilization of rosuvas-tatin were temperature 180OC and heating time of 30min, because of the lowest free radical concentra-tion.

The parameters of the EPR spectra of heatedrosuvastatin changed with microwave power of themeasurement. The influence of microwave power inthe range from 2.2 mW to 70 mW on amplitudes (A)of the EPR lines of rosuvastatin thermally sterilizedat temperatures: 160OC (120 min), 170OC (60 min)and 180OC (30 min), are presented in Figure 6 a-c,respectively. Amplitudes (A) of the heated drugsamples increased with increasing of microwavepower (M/Mo) and they were saturated in the testedrange of microwave power. Such correlation was

Figure 5. Comparison of free radical concentrations (N) in rosuvastatin thermally sterilized at temperatures 160OC during 120 min, 170OCduring 60 min and 180OC during 30 min

Figure 6. The influence of microwave power on amplitudes (A) of the EPR spectra of rosuvastatin sterilized at temperatures: 160OC (120min), 170OC (60 min), and 180OC (30 min). M ñ microwave power used during the measurement of the EPR spectra, Mo ñ total microwavepower produced by klystron (70 mW)


Figure 7. The influence of microwave power on linewidths (∆Bpp) of the EPR spectra of rosuvastatin sterilized at temperatures: (a) 160OC(120 min), (b) 170OC (60 min) and (c) 180OC (30 min). M ñ microwave power used during the measurement of the EPR spectra, Mo ñ totalmicrowave power produced by klystron (70 mW)


characteristic for low spin-lattice relaxation process-es (10) in all the tested samples. It indicated thattemperature of heating did not change magneticinteractions between free radicals and molecularstructure in rosuvastatin.

The influence of microwave power from 2.2mW to 70 mW on linewidths (∆Bpp) of the EPR spec-tra of rosuvastatin heated at temperatures: 160OC(120 min), 170OC (60 min) and 180OC (30 min), arepresented in Figure 7 a-c, respectively. The increaseof linewidths (∆Bpp) with increasing of microwavepower was observed for all the examined rosuvas-tatin samples. The broadening of EPR lines withincreasing of microwave power was characteristicfor homogeneous broadening of EPR lines (10-12).

The product of thermal sterilization of rosuvas-tain at temperature 180OC (30 min) was expected asthe drug with the highest safety of application inpatient. It contained the relatively lower free radicalconcentration compared to the rosuvastatin thermal-ly sterilized at temperatures 160OC (120 min) and170OC (60 min). Free radicals introduced to the tis-sues may react with their molecules and change theirchemical structures, which may be accompanied bynegative effects (13-15). Free radicals may initiateperoxidation of lipids, they may destroy cell mem-branes or modify chemical units in cells (13-15).Free radicals of sterilized rosuvastatin may reactwith o-semiquinone free radicals in melaninbiopolymers in human organism and the interactionof this drug will be prolonged. Interactions of drugswith free radicals of melanins via EPR examinationwere described for example in papers (16-18). Freeradicals play an important negative role in majorarteriosclerosis, diabetes, arthrosis, cataract,nephropathy, cancer (13-15). It is expected that suchnegative effects will be the lowest during therapy byrosuvastatin sterilized at temperature 180OC during30 min.

The performed electron paramagnetic reso-nance investigation of rosuvastatin bring to lightparamagnetic character of thermally sterilized sam-ples of rosuvastatin. Free radicals were responsiblefor the EPR spectra of heated rosuvastatin. The opti-mal temperature and time of thermal sterilization ofrosuvastatin were determined. The results may bepractically used during production of rosuvastatin.

CONCLUSIONS

EPR investigation of rosuvastatin thermallysterilized at temperatures 160OC during 120 min,170OC during 60 min and 180OC during 30 minpointed out that:

Thermal sterilization produced free radicals inrosuvastatin with concentration decreasing withheating temperature, and the best conditions forthermal sterilization of rosuvastatin resulting withthe lowest free radical concentration were tempera-ture 180OC and heating time of 30 min.

The influence of microwave power on EPRspectra indicated that slow spin lattice relaxationprocesses existed in the heated rosuvastatin and thelines were homogeneously broadened.

Usefulness of electron paramagnetic resonancespectroscopy to optimization of thermal sterilizationconditions for rosuvastatin was confirmed.

Acknowledgment

This work was financially supported byMedical University of Silesia in Katowice, grantnumber: KNW-1-076/K/5/0

Conflict of interests

The authors declare that there is no conflict ofinterests regarding the publication of this paper.They have no conflict of interests or no financialgains in mentioning the company names or trade-marks. The authors of the paper do not have anyfinancial relation with the commercial identity men-tioned in our paper.

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tured under aseptic conditions. PolishCommittee for Standardization, Warszawa2002 (in Polish).

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Received: 20. 07. 2015


The medicinal value of plants used in phy-totherapy depends on the quality of habitats fromwhich they are obtained. The dependence betweenenvironmental pollution and plant contaminationwith heavy metals is stressed in the literature (1, 2).Also the capacity of medicinal plants to accumulatemetals was indicated by many authors (2-4).Considering the quality of herbal raw material usedin phytotherapy, the place of a plant origin is veryimportant. Leaf material is usually unwashed,despite the fact that the above-ground parts of plantsare exposed to dust precipitation, including fine dust,particularly enriched with heavy metals, such as: Cu,Zn, Mn and Fe (4-7). Thus, the evaluation of select-ed metal content in the unwashed and washed leavesof plants used in phytotherapy will allow to answerthe question whether atmospheric precipitation canbe an additional metal enrichment source. Urticadioica L. (common nettle) and Plantago major L.(plantain) were two plant species chosen for thestudy. The common nettle represents the Urticaceaefamily, genus Urtica; the plantain represents the

Plantaginaceae family, genus Plantago. Both thecommon nettle (8) and the broadleaf plantain (9)have a wide spectrum of use in phytotherapy. Thesetwo species differ, among others, in the structure oftheir above-ground parts and root morphology.Urtica dioica L. has a long and straight single stemwith hairy stinging leaves. It grows up to the heightof 1.2 m. Plantago major L. reaches the height ofabout 0.4 m (with florescence), and is characterizedby a short hypophyllous stem. The nettle has a tap-root with numerous sarmenta in the top section of theroot neck. The broadleaf plantain has a ramifiedfibrous root system. Both species prefer fertilesynanthropic habitats and are easy to identify. Theyare widespread in Europe, Asia and America.Samples used in the present study were collectedfrom an area in Eastern Poland (Europe) and the lev-els of microelements, such as: Cu, Zn, Mn and Fe,were identified. These metals have significant meta-bolic functions in the human body and they are oftenstudied in herbal plants (e.g., 10-14). It should beemphasized that in the literature related to the accu-

TRACE METAL CONTENT (Cu, Zn, Mn AND Fe) IN URTICA DIOICA L. AND PLANTAGO MAJOR L.

ELØBIETA KR”LAK1*, JOLANTA RACZUK1 and LIDIA BORKOWSKA2

1 Department of Ecology and Environmental Protection, Institute of Biology, Siedlce University of Natural Science and Humanities, 08-110 Siedlce, Poland

2 Department of Botany, Institute of Biology, Siedlce University of Natural Science and Humanities,Siedlce, 08-110 Siedlce, Poland

Abstract: The aim of the study was to compare the contents of Cu, Zn, Mn and Fe in the washed and unwashedleaves and roots of two plant species: Urtica dioica L. and Plantago major L., used in herbal medicine. Thesetwo herb species occur in the same environmental habitats, yet their morphological structure is different. Thesoil and plant samples for analyses were collected from an uncontaminated area in Eastern Poland. In each habi-tat location, the samples were taken from sandy soils with slightly acidic and neutral pH values. The obtainedresults showed that U. dioica and P. major accumulated similar amounts of trace metals, such as: Cu, Zn andFe, in leaves, despite the differences in the morphological structure of their overground parts. The content ofMn in leaves U. dioica was about twice as much as in P. major. Also, no differences in the metal content wereobserved between washed and unwashed leaves of both species. However, in the same habitat conditions, a sig-nificantly higher content of Cu, Zn and Mn was found in the roots of P. major than U. dioica. The content ofFe in the roots was similar in both species. P. major and U. dioica may be a valuable source of microelements,if they are obtained from unpolluted habitats.

Keywords: leaves, roots, herbal medicine, soil, heavy metals, enrichment coefficient

1447

* Corresponding author: e-mail: [email protected]; phone: +48256431217; fax: +48256445959

1448 ELØBIETA KR”LAK et al.

mulation of selected metals in Urtica dioica L. andPlantago major L. occurring in the same habitats,there are no data from Eastern Poland.

Main research hypotheses in the study:● the contents of metals: Cu, Zn, Mn and Fe in

unwashed leaves of Urtica dioica and Plantagomajor are higher than in washed leaves,

● metals accumulation in morphological parts(leaves, root) depends on the plant species,

● significant correlations exist between the metalcontent in soil and metal content in the leaves androots of the analyzed plant species.

The scope of the study:● analysis of soil properties at the plant collection

sites: pH, organic carbon and nitrogen content,granulometric composition, the content of Cu, Zn,Mn and Fe,

● determination of the content of Cu, Zn, Mn andFe in the unwashed and washed leaves and in theroots of both plant species.

MATERIALS AND METHODS

Experimental material collection

The area in Eastern Poland (Europe) locatedamong the following towns: Bia≥ystok (23O10íE,53O08íN), Ostro≥Íka (21O35íE, 53O05íN), Garwolin(21O38íE, 51O54íN) and Bia≥a Podlaska, (23O07íE,52O02íN) was chosen for the study. Within the area,15 research sites were randomly selected. Soil sam-ples and plant material were collected in the spring(May/June) of 2013, prior to blooming. At eachsampling site, an area of approximately 2,000 m2

was selected for the experiments. A special care wastaken to choose the sites where the broadleaf plan-tain and the common nettle occurred in close prox-imity. Soil samples were collected from the surfacesoil layer at the depth of 0-15 cm with the use of theEgner soil sampler. Ten soil subsamples were col-lected from each site. The soil was subsequently air-dried in the laboratory and passed through a sieve of2 mm mesh. The soil samples were stored in closedPE containers pending further analyses.

Ten individual plants of each species (Urticadioica and Plantago major) were collected from eachsite in the chosen area. The plant material was care-fully cleaned of the soil and both leaves and roots wereseparated from the stem. The plant leaves collectedfrom each site were divided in half and placed in bags.One of the leaf subsamples of each species was care-fully washed in distilled water. A similar procedurewas applied in the case of the roots. Subsequently, allthe subsamples (washed and unwashed), as well as theplant roots, were dried at the temperature of 60OC until

obtaining dry matter (d.m.). The dried plant materialwas homogenized in a vibration mill equipped with atungsten carbide bowl.

Experimental material analyses

Soil samples were tested for their granulometriccomposition with the use of the Casagrandeísmethod. Soil pH was measured with a glass electrodepH-meter, 1 : 2.5 (soil : 1 M KCl solution) and organ-ic carbon was determined with the Tiurin method(15). Soil samples (0.3000 g) were mineralized inKiejdahlís flasks using 95% H2SO4 and 30% H2O2 (3: 1, v/v). The total soil nitrogen content was deter-mined with the phenyl hypochlorite method (16).

Soil, leaves and roots samples (1.0000 g)underwent microwave mineralization using 65%HNO3 and 30% H2O2 (5 : 3, v/v). After wet mineral-ization, contents of metals: Cu, Zn, Mn and Fe, weredetermined with the AAS technique (Carl Zeiss JenaAAS 30).

Each sample was analyzed four times usingpure reagents. The quality of the analytical proce-dure was checked using a reference material(Certified Reference Material CTA-OTL-1 OrientalTobacco Leaves, Department of AnalyticalChemistry, Institute of Nuclear Chemistry andTechnology, Poland).

Statistical analysis

The results of the analyses were statisticallycompared. The profile of the measurement data dis-tribution was checked using the Shapiro-Wilk test.The similarity of the physical and chemical proper-ties of the soils was compared by means of the clus-ter analysis. The t-Student test was applied to com-pare the results of the metals content in unwashedand washed leaves and in unwashed leaves and rootsof the plants. Correlations between soil propertiesand metals content in the analyzed morphologicalparts of the plants were investigated using Pearsoníslinear correlation coefficient. Statistical tests wereperformed using Statistica software (version 10).Metal accumulation capacity of the morphologicalplant parts was determined by calculating theenrichment coefficient (EC). For this purpose, thefollowing ratios were used: EC = metals content inplants (leaves, roots)/metals content in soil.


Analyzed physical and chemical soil propertieswere highly similar in the research sites, whichmight be confirmed by a close Euclidean distance(Fig. 1). The analyzed soils showed slightly acidic

Trace metal content (Cu, Zn Mn, and Fe) in Urtica dioica L. and Plantago major L. 1449

and neutral reactions. They were relatively rich inorganic carbon and total nitrogen (Table 1). Thesites selected for the study were primarily represent-ed by light sandy soils (sites: 1, 2, 4, 6, 12, 14 ñweakly loamy sand, 3, 7-11 ñ loamy sand). Thesandy fraction (2.0 ñ 0.05 mm) content in the othersites exceeded 75%. Only two sites (5 and 13) hadsoils with a granulometric composition correspon-ding to light loam, characterized with a lower con-tent of sandy fractions of 63 and 68%, respectively. The metals content in the analyzed soils had the fol-lowing pattern: Cu < Zn < Mn < Fe. The mean con-tents were: 6.947 ± 3.052 mg/kg d.m. for copper,

59.14 ± 25.64 mg/kg d.m. for zinc and 134.7 ± 33.42mg/kg d.m. for manganese. The mean iron contentwas 4.287 ± 1.595 g/kg d.m.

A wide range of the examined physicochemi-cal properties (Table 1) shows that the test plantswere collected from different habitats characterizedby the multiple parameters. This was also confirmedby the standard deviation and range of metals foundin the studied tissues of plants (Table 2). The accu-racy of the AAS method used for the metal determi-nation was confirmed by the results of referencematerial analysis (CRM CTA-OTL-1). According tothe certificate, the metal content in the reference

Table 1. Chosen physical and chemical properties of soils.

Parameters Unit Mean ± SD Range

Content of sand fraction (2-0.05 mm) 78.4 ± 7.41 63-90

Content of silt fraction (0.05-0.002 mm) % 16.5 ± 5.41 8-26

Content of clay fraction (< 0.002 mm) 1.8 ± 0.62 1-11

Acidity pH (in 1 M KCl) 6.62 5.61-7.29

Organic Cg/kg

26.03 ± 12.66 10.92-61.02

Total N 1.833 ± 0.647 1.044-2.898

C/N 13.9 ± 0.308 10.04-21.2

Table 2. The mean content (± SD) and range of results of trace metal analysis in Urtica dioica (Ud) and Plantago major (Pm).

Metal Unit Species of plant Unwashed leaves Washed leaves Roots

Ud 10.67 ± 3.78 10.21 ± 2.83 7.012c ± 1.74

(3.20-15.4) (6.37-17.4) (4.29-10.40)Cu

Pm 8.04 ± 2.51 7.94 ± 2.68 15.16d ± 4.08

(4.38-12.19) (4.30-13.86) (9.95-23.81)

Ud 51.90 ± 25.37 49.21 ± 11.39 61.51c ± 32.26

(13.9-111.9) (27.97-75.8) (33.4-148.7)Cu

Pm 58.80 ± 21.78 58.70 ± 22.57 137.30d ± 59.97mg/kg

(26.46-109.9) 30.58-107.1) (54.9-263.3) dry mass

Ud 59.52a ± 28.67 57.32a ± 17.62 39.55c ± 14.83

(12.2-130.1) (25.7-85.8) (24.6-84.9)Mn

Pm 28.13b ± 14.37 26.14b ± 12.70 76.92d ± 51.68

(14.6-65.2) (9.64-57.7) (19.8-195.1)

Ud 372.00 ± 210.60 286.20 ± 97.67 271.80 ± 159.70

(121.3-712.7) (131.5-454.1) (67.8-640.3)Fe

Pm 345.40 ± 221.70 218.40 ± 97.03 255.20 ± 424.10

(131.6-891.6) (94.2-379.0) (62.7-1775.0)

Superscripts a, b, c and d indicate statistically significant differences in the element content of different parts of the tested plant species.


sample (ppm = mg/kg) was as follows: Cu ñ 14.1,zinc ñ 49.9, Mn ñ 412.0 Fe ñ 989.0. In the presentstudy, the following results were obtained (mg/kg):Cu - 15.42 ± 1.66, Zn ñ 50.4 ± 1.81, Mn - 436.7 ±22.11, Fe - 949.2 ± 34.6.

The contents of Cu, Zn, Mn and Fe in theunwashed nettle leaves did not significantly differfrom the content of these metals in the washedleaves. Likewise, no differences were foundbetween the content of these metals in the unwashedand washed leaves of the broadleaf plantain.Considering the lack of differences between the con-tents of the above metals in the unwashed andwashed leaves, further comparisons referred to theresults of the metals content analyses in theunwashed leaves. This approach results from the

fact that only unwashed leaves are used for phy-totherapeutic purposes. Among the analyzed metals,more Mn (t28 = 3.79, p < 0.001) was accumulated bythe nettle leaves in comparison to the plantainleaves. The mean manganese content of theunwashed nettle leaves was approximately twice ashigh as that of the plantain leaves, amounting toapproximately: 59 and 28 mg Mn/kg d.m. The othermetals (Cu, Zn, Fe) were accumulated in similaramounts in the unwashed nettle and plantain leaves(Table 2).

Cu, Zn and Mn content in the broadleaf plan-tain roots were about twice as high as the amounts ofthese metals in nettle roots (the tested statistic para-meters had the following values: Cu: t28 = ñ 7.122, p < 0.001, Zn: t28 = ñ 4.311, p < 0.001 and Mn:

Figure 1. Dendrogram similarity of research sites

Figure 2. Metal enrichment coefficient in the leaves of Urtica dioica (Ud) and Plantago major (Pm). The superscripts a and b indicatestatistically significant differences


t28 = ñ 2.692, p = 0.012). No differences were foundbetween the iron contents in the nettle and broadleafplantain roots (Table 2).

The enrichment coefficients (EC) in the leavesof both plant species attained values > 1 for Cu andZn, and < 1 for Mn and Fe (Figs. 2 and 3).Significant differences in EC values for the leaf/soilsystems of both species were found only for Mn (t28

= 3.135, p = 0.004) (Fig. 2). Also, significant differ-ences in EC values were observed between theroot/soil systems for Cu (t28 = ñ 3.717, p < 0. 001),

Zn (t28 = ñ 2.756, p = 0.010) and Mn (t28 = ñ 2.264,p = 0.031) (Fig. 3).

Significant correlations (p < 0.05) were foundbetween the contents of the following metals in soil:Cu and Zn (r = 0.654), Cu and Fe (r = 0.705), Cu andMn (r = 0.545), Zn and Mn (r = 0.630). Correlations(p < 0.05) were also observed between the coppercontents in the leaves and roots of the broadleaf plan-tain (r = 0.879) and between the zinc contents in theleaves and roots of the broadleaf plantain (r = 0.662).Such interrelations were not identified in the case of

Table 3. The content of trace metals in leaves (L) and roots (R) of the analyzed plants and in soil (S) according to literature.

Plant Sample Cu Zn Mn Fe Area of study

mg kg-1 (reference)

L 13-41 14-152 29-78

R 15-106 26-210 86-182 Roma/Italy (27)

S 26-186 92-428 496-626

Urtica L 2.9 14.6 51 342 Territory of Fergana/Uzbekistan (13)

dioica L. L 13.23 29.14 57.84 303 Territory of Novi Sad/Serbia 14)

R 12.71 22.75 28.12 673

L 12.27 21.44 11.96 Skopje region/Macedonia (26)

R 9.58 19.55 9.94

L 10-60 39-121 21-92

R 20-124 37--212 48-171 Roma/Italy (27)

S 27-214 71-368 449-784

Plantago L 12 22 260 380 Territory of Fergana/Uzbekistan (13)

major L. L 12.4 80 69 DureÎs region/Albania (1)

S 34.6 259.1 172

Figure 3. Metal enrichment coefficient in the roots of Urtica dioica (Ud) and Plantago major (Pm). The superscripts a and b indicate sta-tistically significant differences


the nettle. No correlations were found between themetal contents in the leaves and roots of Urtica dioicaand Plantago major and the metal levels in soil.

The plant species selected for the study: Urticadioica and Plantago major, prefer synanthropic habi-tats, characterized by the same edaphic parameters,e.g.: soil trophism, acidity, organic matter contentand granulometric composition (17). The analyzedplants were found on sandy and sandy and clayeysoils, typical of north-eastern Poland. The metal con-tents identified in the surface soil layers remain with-in the value ranges determined for the sandy soils ofPoland, amounting to [mg/kg]: Cu: 1-26, Zn: 7-150,Mn: 15-1500 and Fe: 3.2-10.7 g/kg (18).

The metal content of natural soils is closelyconnected with the chemical composition of the par-ent rock. The metal contents in the parent rock wereassumed as the geochemical background for the soilin a given area (19). The copper content analyzed inthe surface soil layer in the studied area remainedwithin the value range (19) as the geochemical back-ground, at the level of 7.1 mg/kg. The zinc value, inturn, was slightly higher in comparison with theconcentration of 30 mg/kg determined as the back-ground. The principal source of metals in the regionare energy-related processes connected with thecombustion of solid fuels, mainly coal, in the heat-ing season (October - March).

The atmospheric emissions of fine-graineddust fractions from energy-related processesinclude, e.g., Zn, Cu, Mn, and Fe (4-7, 20, 21).Virtually no constant sources of atmospheric pollu-tion exist during the growing season in the region,when the plants are collected for phytotherapeuticpurposes. Thus, atmospheric dust settles on the leafsurface in negligible amounts. This is confirmed bythe lack of significant differences between the con-tents of Cu, Zn, Mn and Fe in the unwashed andwashed leaves of both the nettle and broadleaf plan-tain. However, the results of metal content analysisin the unwashed leaves, compared with washedleaves, showed a larger standard deviation in themajority of cases.These results show that the area ofEastern Poland has favorable environmental condi-tions for obtaining plants for the purposes of phy-totherapy, without the risk of contamination with,e.g., heavy metals, such as Cu and Zn.

Although the plants were collected from thesites with similar physical and chemical soil proper-ties, the metal contents analyzed in the selected mor-phological plant parts revealed interspecies differ-ences. Definitely more Cu, Zn and Mn was identifiedin the roots of the broadleaf plantain than in the net-tle. No significant differences were found between

the contents of Cu, Zn and Fe in the plantain and net-tle leaves. In the case of manganese, in turn, signifi-cantly larger amounts were accumulated in theleaves of the nettle in comparison with the plantain.

The principal transport path for metals fromsoil to plants is the root system. The plantain has afibrous root system, whereas the nettle ñ a taproot.Thus, the plantain has a larger contact area betweenthe root surface and soil in comparison with the net-tle. The intake of metals by plant roots is determinedby specific conditions in the rhizosphere, includingthe presence of microorganisms that activate soilenzymes (22, 23). The relationship between thelevel of root ramification and the soil enzyme activ-ity in the rhizosphere was indicated by Hedley et al.(24). The soil enzyme activity in the rhizosphere isincreasingly higher if the plant root system is moreelaborate. The multiplicity of small root ramifica-tions ensures active water and nutrient intake by theplants, and the root surface is the accumulation sitefor selective soil microflora (25). The obtainedresults show that, in the same environmental condi-tions, the plantain roots accumulate more Cu, Znand Mn than the nettle roots; in the case of phy-totherapeutic use, the former are potentially agreater source of microelements than the nettleroots. On the other hand, Urtica dioica leaves con-tain more manganese than Plantago major leaves.

The metal contents detected in this study in theleaves and roots of the nettle and plantain remain with-in the value range cited in the literature (13, 14, 26)(Table 3). The experimental material came from non-polluted areas (13, 14, 26). Higher Cu, Zn and Mncontents than in the present study were identified inthe leaves and roots of the nettle and plantain (27), col-lected as research samples in the area of Rome (Table3). Higher Cu, Zn and Mn contents in the leaves ofPlantago major were also determined (1) in the terri-tory of Albania. It should be noted that the area wasmore polluted with heavy metals than the area select-ed for the present study, with the values of Cu and Znenrichment coefficients in the analyzed morphologicalplant parts above (1, 27). The enzymatic activity of thesoil decreases along with soil pollution with heavymetals, resulting in a lower metal accumulation coef-ficient in the plant tissues (28).

Some authors (1, 27) point to the correlationsbetween the metals content of the soil and morpho-logical parts of the plants. The results obtained in thepresent study do not confirm the reported data. Nosignificant correlations were found between the met-als content of the soil and analyzed plants. Themobility and availability of heavy metals for plantsdepends on, e.g., granulometric composition, soil


reaction and organic matter content (23). Thus, itcan be assumed that in soils similar to those fromwhich Urtica dioica and Plantago major wereobtained for the study, metals accumulation is deter-mined by the physiological demand of the plants forparticular elements.

The obtained results on metal accumulation inthe leaves and roots of Urtica dioica and Plantagomajor in the analyzed area can serve as referencedata for other regions more exposed to contaminantemission from anthropogenic sources than the ana-lyzed one. On the world scale, these regions includeAsia, from which was evaluated the largest amountsof trace metals from anthropogenic sources emittedto the atmosphere in connection with the rising glob-al demand for electricity (29).

CONCLUSION

The results of the study revealed no significantdifferences between the contents of the studied met-als in the unwashed and washed leaves of both thenettle and the broadleaf plantain. They suggest anegligible impact of atmospheric precipitation onthe surface deposition of metals on the lamina.

In the same habitat conditions, greater amounts ofCu, Zn and Mn are accumulated in the roots of thebroadleaf plantain than in the common nettle roots; thiscan result from differences in the root system structureof both plants. On the other hand, despite the differ-ences in the morphological structure of their above-ground parts, Cu, Zn and Fe contents in the leaves ofUrtica dioica and Plantago major were similar.

Urtica dioica and Plantago major obtainedfrom synanthropic habitats, characterized withsandy soils with slightly acidic and neutral pH val-ues and uninfluenced by constant sources of heavymetal supply, can be used in herbal medicine with-out running the risk of plant contamination withsome heavy metals.

REFERENCES

1. Bekteshi A., Bara G..: Pol. J. Environ. Stud. 22,1881 (2013).

2. Belabed S., Lotmani B., Romane A.: J. Mater.Environ. Sci. 5, 1551 (2014).

3. Sembratowicz I., Rusinek E., Ognik K.,TruchliÒski J.: Herba Pol. 55, 22 (2009).

4. Jena V., Gupta S.: J. Environ. Anal. Toxicol. 2,161 (2012).

5. Kowol J., KwapuliÒski J., Brodziak-Dopiera≥aB., Paukszto A., Bogunia M. et al.: Pol. J.Environ. Stud. 20, 115 (2011).

6. FilipoviÊ-TrajkoviÊ R., Zoran S., IliÊ Z.S.,Ljubomir äuniÊ L., AndjelkoviÊ S.: J. FoodAgric. Environ. 10, 959 (2012).

7. Schreck E., Foucault Y., Sarret G.., SobanskaS., CÈcillon L. et al. C.: Sci. Total Environ. 427,253 (2012).

8. Joshi B.C., Mukhija M., Kalia A.N.: Int. J.Green Pharm. 8, 201 (2014).

9. Samuelsen A.B.: J. Ethnopharmacol. 71, 1(2000).

10. Basgel S., Erdemo�lu S.B.: Sci. Total Environ.359, 82 (2006).

11. Pytlakowska K., Kita A., Janoska P., Po≥owniakM., Kozik V.: Food Chem. 135, 494 (2012).

12. Soylak M., Cihan Z., Yilmaz E.: Environ.Monit. Assess. 184, 3455 (2012).

13. Igamberdieva P.K., Danilova E.A.: Global J.Biotechnol. Biochem. 8, 66 (2013).

14. Mihaljev Z., Zivkov-Balos M., CupiÊ Z. JaksiÊS.: Acta Pol. Pharm. Drug Res. 71, 385 (2013).

15. Ostrowska A., GawliÒski S., Szczubia≥ka Z.: Inst.Environ. Prot. Press, (in Polish), Warszawa 1991.

16. Solorzano L.: Limnol. Oceanogr. 14, 799(1969).

17. Zarzycki K., TrzciÒska-Tacik H., RÛøaÒski W.,Szelπg Z., Wo≥ek J., Korzeniak U.: W. SzaferInstitute of Botany, PAS, KrakÛw 2002.

18. Kabata-Pendias A., Pendias H.: Biogeochemis-try of trace elements (in Polish) PWN, War-szawa 1999.

19. Czarnowska K.: Roczn. Glebozn. 47, 43 (1996).20. Linak W.P., Miller C.A., Wendt J.O.L.: J. Air

Waste Manage. Assoc. 50, 1532 (2000).21. Manousakas M., Eleftheriadis K., Papaefthy-

miou H.: Aerosol Air Qual. Res. 13, 804 (2013). 22. Khan S., Cao Q., Hesham A.E.L., Xia Y., He J.:

J. Environ. Sci. 19, 834 (2007).23. Wyszkowska J., Borowik A., Kucharski M.,

Kucharski J.: J. Elem. 18, 769 (2013).24. Hedley M.J., Nye P.H., White R.E. New

Phytologist 95, 69 (1983).25. McCully E.: Annu. Rev. Plant Physiol. Plant

Mol. Biol. 50, 695 (1999).26. Rafajlovska V., Kavrakovski Z., Simonovska J.,

Srbinoska M.: Qual. Life Res. 4, 26 (2013).27. Malizia D., Giuliano A., Ortaggi G., Masotti A.:

Chem. Cent. J. S6, 1 (2012).28. Lee I.S., Kim O.K., Chang Y.Y., Bae B., Kim

H.H., Baek K.H.: J. Biosci. Bioeng. 94, 406(2002).

29. Pacyna J.M., Pacyna E.G..: Environ. Rev. 9,269 (2001).

Received: 12. 10. 2015


2,3-Diphenylcyclopropenone (DCP) is a topi-cally administered agent intended primarily fortreating alopecia areata (AA). AA is a T cell-medi-ated autoimmune disease involving hair folliclescharacterized by hair loss. Frequency of AA rangesfrom 0.7% to 3.8% of patients attending dermatol-ogy clinics (1). Etiopathogenesis of AA is notknown but evidence exists to support genetic,immune and environmental factors (2, 3). DCPplays the role of a sensitizing agent. It is also beingused as an immune-modulating therapeutic factor.Although the mechanism of action of DCP has notbeen clearly defined, DCP induces changes in theperifollicular CD41/CD81 T-lymphocyte ratio (4),apoptosis of perifollicular lymphocytes (5) andmodulate proinflammatory cytokines (6). Recently,there are numerous studies suggesting DCPís goodefficiency in treatment of cutaneous metastatic

melanoma (7, 8) suggesting the growing importanceof this potential drug. Unfortunately, very little isknown about the pharmacokinetics of DCP. The aimof this study was to investigate the stability of DCPin an environment similar to human body fluids or inthe situation of contamination DCP samples withwater. For this purpose the authors have chosen,respectively, 0.9% sodium chloride aqueous solu-tion and water that was added to DCP solutions.

EXPERIMENTAL

Materials and Methods

The authors chose solutions of DCP in propy-lene glycol because these were used successfully intheir previous studies regarding treatment of AA (8).Usually, the concentration of 3% DCP is being usedduring the process of sensitization, at the beginning

STUDIES ON STABILITY OF 2,3-DIPHENYLCYCLOPROPENONE IN CONTACT WITH WATER AND AQUEOUS NaCl SOLUTIONS.

CONCLUSIONS FOR PURPOSE OF TOPICAL THERAPY OF PATIENTS WITH ALOPECIA AREATA

TOMASZ WASY£YSZYN1* and KATARZYNA BOROWSKA2,3

1Medical Center GABINET DERMATOLOGICZNY TOMASZ WASY£YSZYN, 25/421 Gen. Anders St., 00-159 Warszawa, Poland

2Medical Center CADERM, 87/113 Marsza≥kowska St., 00-683 Warszawa, Poland 3Department of Histology and Embryology with Experimental Cytology Unit, Medical University of Lublin,

11 Radziwi≥owska St., 20-080 Lublin, Poland

Abstract: Diphenylcyclopropenone (DCP) is a topically administered agent used for more than three decadesfor treatment of alopecia areata (AA). Moreover, numerous recent studies show itís efficiency in treatment ofcutaneous metastatic melanoma. Despite being a potentially useful drug still very little is known about the phar-macokinetics of DCP. The authors investigated the stability of DCP solutions in propylene glycol with the addi-tion of 0.9% aquous solution of natrium chloride (0.9% NaCl) or with water. DCP was prepared in two con-centrations: 0.1% and 3%. Itís stability was then measured with different proportions of 0.9% NaCl or wateradded and in different temperatures. Contrary to common opinion that DCP solutions are extremely unstable,authors have found them to be relatively stable. DCP solutions with the addition of equal quantity of 0.9% NaCldecomposed slowly at the temperature of 37∞C but after 70 h all solutions still contained more than 80% of ini-tial DCP. Solutions of DCP with the addition of 1% of water at the temperature of 4∞C are in the present studystable, containing more than 98% of initial DCP after 20 days. Authors discuss the results in spite of possiblemetabolism of DCP on the surface of human skin during topical immunotherapy.

Keywords: alopecia areata, contact sensitizers, diphenylcyclopropenone, stability, topical immunotherapy, sol-vents

1455


1456 TOMASZ WASY£YSZYN and KATARZYNA BOROWSKA

of the treatment. Later on, smaller concentrations areadministered to maintain moderate allergic reactionto DCP (8) and most often it is 0.1%. Hence, thesetwo concentrations were chosen for this study. It wasarranged to mimic three situations found during top-ical treatment with DCP: the treatment with 0.1%DCP (A), the sensitization with 3% DCP (B) and thepotential contamination of medication with a smallamount of water (C). The authors made three seriesof measurements, respectively: 0.1% DCP with 0.9%NaCl in proportions 1 : 1 (A), 3% DCP with 0.9%NaCl in proportions 1 : 1 (B) and 3% DCP with 1%addition of water which means proportions of 99 : 1(C). The first two series of measurements have beenperformed at the temperature of 37OC as it resemblesthe temperature of the human body. These weremeasured up to 70 h, as the therapeutic solutions ofDCP (both 0.1% and 3%) are being washed from thescalp after 24-70 h. The last one was made at thetemperature of 4OC like in the refrigerator where the

DCP preparations are stored. This one was observedup to 20 days as authors usually prepare new DCPsolutions once every 2-3 weeks. The study was per-formed for the standard of diphenylcyclopropenone(No. 177377, Sigma Aldrich) dissolved in propyleneglycol (No. W294004, Sigma Aldrich) and in 0.9%NaCl aqueous solution. Samples were analyzed byhigh performance liquid chromatography with diodearray detection (HPLC-UV DAD) using internal nor-malization method.

Method description: HPLC-DAD conditions:column: Phenomenex C18, 250 mm ◊ 4.6 mm; oventemperature: 37OC; mobile phase: acetonitrile +water (80 + 20); mobile phase flow: 1 mL/min; λ =296 nm. Reagents: diphenylocyclopropenone(DCP), 99%; Alfa Aesar, batch No. 10130265;diphenylacetylene (DPA); 98%, Aldrich, batch No.STBC7355V; propylene glycol, Sigma-Aldrich;deionized water, ultra pure, Millipore; acetonitrile,99.9%, POCh; sodium chloride, POCh.

Table 1. Decomposition of 3% DCP solution in propylene glycol (without additions). Numbers show propor-tions of DCP (left side) to it's product of decomposition, diphenylacetylene - DPA (right side).

Time

0 After 7 days After 14 days Temperature

99.89 / 0.11 99.89 / 0.11 99.88 / 0.12 + 4OC

99.87/ 0.13 99.84 / 0.16 97.62 / 2.38 + 37OC

Table 2. 0.1% DCP solution in propylene glycol with the addition of 0.9% NaCl in proprtions 1 : 1, v/v.Amount of DCP shown in percentage of the initial amount at time "0". Temperature + 37OC.

Time of Area of DCP percentagemeasurement DCP sample according to time C [mmol/L]

[h] (mV ◊ min) "0"

0 9222757 100.0 4.85

4 9120211 98.9 4.80

5 8290834 89.9 4.36

8 7891536 85.6 4.15

11 8041933 87.2 4.23

15 7976472 86.5 4.19

18 8053455 87.3 4.23

28 7966278 86.4 4.19

35 7873213 85.4 4.14

44 7965499 86.4 4.19

51 7969064 86.4 4.19

70 7964593 86.4 4.19

Note: Area of DCP sample (Tables 2-5) is area under the curve, measured as a product of peak's height in mili-volts and it's length in minutes.

Studies on stability of 2,3-diphenylcyclopropenone in contact with water and... 1457

Analysis of DCP stability at a concentration level

of 3% dissolved in propylene glycol (no addi-

tions)

There were two 3% DCP mixtures in propy-lene glycol: stored at 37OC and 4OC. In determinedtime intervals (after 7 and 14 days) the solution wasanalyzed using HPLC technique. Peak area of DCPmeasured at time zero (at the beginning of the exper-iment) was compared with data obtained after abovementioned storage period intervals.

Analysis of DCP at a concentration level of 0.1%

dissolved in propylene glycol and in 0.9% NaCl

aqueous solution

DCP at a concentration level of 0.1% was pre-pared by dissolving the standard of DCP in a mixtureof propylene glycol and 0.9% aqueous NaCl (1 : 1,v/v). The obtained solution was stored at 37OC withlight exposure. At determined time intervals (after 4, 5,8, 11, 15, 18, 28, 35, 44, 51 and 70 h) the solution wasanalyzed using HPLC technique. Peak area of DCP

Table 3. The amount of diphenylacetylene (DPA) in 0.1% DCP solution in propylene glycol with the additionof 0.9% NaCl in proportions 1 : 1, v/v, using the same scale as in Table 2. Here 100% would mean that solu-tion contains 0.1% DPA and all DCP has been decomposed. Temperature + 37OC.

Time of Area of DPA percentagemeasurement DPA sample according to time C [mmol/L]

[h] (mV ◊ min) "0"

0 42810 0.46 0.026

4 47859 0.52 0.029

5 842408 9.22 0.517

8 1195831 13.16 0.738

11 1241483 13.37 0.750

15 1224767 13.31 0.747

18 1245381 13.39 0.751

28 1240928 13.48 0.756

35 1220030 13.42 0.753

44 1227427 13.35 0.749

51 1241313 13.48 0.756

70 1246547 13.53 0.759

Table 4. 3% DCP solution in propylene glycol with the addition of 0.9% NaCl in proprtions 1 : 1 v/v. Amountof DCP shown in percentage of the initial amount at time "0" . Temperature + 37OC.


[h] (mV ◊ min) "0"

0 14541447 100.0 145.46

4 14612465 100.5 146.19

5 14278356 98.2 142.84

8 14123391 97.1 141.24

11 14337265 98.6 143.43

15 13375591 92.0 133.82

18 13292271 91.4 132.95

28 12809334 88.1 128.15

35 12350644 84.9 123.50

44 11832474 81.4 118.41

51 11829550 81.4 118.41

70 11799764 81.1 117.97


measured at time zero (at the beginning of the experi-ment) was compared with data obtained after the abovementioned storage period intervals. Furthermore, con-tent (percentage) of diphenylacetylene, the maindecomposition product of DCP, was assessed.

Analysis of DCP at a concentration level of 3%

dissolved in propylene glycol and in 0.9% NaCl

aqueous solution

DCP at a concentration level of 3% was pre-pared by dissolving the standard of DCP in mixtureof propylene glycol and 0.9% aqueous NaCl (1 : 1,v/v). The obtained solution was stored at 37OC withlight exposure. At determined time intervals (after 4,

5, 8, 11, 15, 18, 28, 35, 44, 51 and 70 h) the solutionwas analyzed using HPLC technique. The samplewas diluted before injection (0.1 mL of sample in 1mL of acetonitrile) not to exceed the detector linear-ity range for the examined analyte. Peak area ofDCP measured at time zero (at the beginning of theexperiment) was compared with data obtained afterabove mentioned storage period intervals.Furthermore, content of diphenylacetylene, the maindecomposition product of DCP, was assessed intime zero (0.58%) and after 70 h (4.80%).

Analysis of DCP at a concentration level of 3%

dissolved in propylene glycol with addition of 1

mass percent of water

Water content in 3% DCP solution in propy-lene glycol was analyzed using the Karl Fischertechnique. The result (0.063%, n = 4) was below theassumed value 0.1%. For the other part of the exper-iment one mass percent of water was added to thesample. DCP solution was stored at 4OC with nolight exposure. At determined time intervals (after 4,24, 28 and 48 h, 6, 12 and 20 days) the solution wasanalyzed using HPLC technique. The sample wasdiluted before injection not to exceed the detectorlinearity range for the examined analyte. Peak areaof DCP measured at time zero (at the beginning ofthe experiment) was compared with data obtainedafter the above mentioned storage period intervals.Furthermore, content of diphenylacetylene, the maindecomposition product of DCP, was assessed intime zero (0.45%) and after 20 days (0.58%).

Validation of the method

The HPLC method was developed for this analy-sis mainly because gas chromatography (GC) has lim-

Table 5. 3% DCP solution in propylene glycol with the addition of water in proportions 99 : 1, v/v. Amount ofDCP shown in percentage of the initial amount at time "0" . Temperature + 4OC.


[h] (mV ◊ min) "0"

0 h 12015758 100.0 145.46

4 h 11953665 99.48 144.71

24 h 11959071 99.53 144.78

28 h 11850459 98.62 143.45

48 h 11845842 98.59 143.41

6 days 11851573 98.63 143.47

12 days 11800043 98.20 142.84

20 days 11798543 98.19 142.83

Table 6. Analysis of seven individual DCP solutions in propyleneglycol, at the same concentration level, injected into chromatogra-phy column.

SolutionArea of

No.DCP sample(mV ◊ min)

1 12185327

2 12101392

3 12068100

4 12113009

5 12100614

6 12152269

7 12086184

Mean 12115271

S.D. 40333

RSD [%] 0.33

RSD = Relative standard deviation

Studies on stability of 2,3-diphenylcyclopropenone in contact with water and... 1459

ited usage in water solutions. The precision (repeata-bility) was determined by analyzing seven individualDCP solutions in propylene glycol, at the same con-centration level, injected into chromatography column(Table 6). Relative standard deviation (RSD) is 0.33%which proves the methodís precision. This denotesabout reliability of the analytical method, especiallywhen the relative concentrations of analytes are pre-sented as results (internal normalization method). Thechromatographic system was stable during the experi-ment. For the long-term analyses, the quality control(QC) standards where used to check the system stabil-ity before injection of DCP solution after storage peri-od. The study was conducted in accredited analyticallaboratory - Institute of Industrial Organic Chemistry,Warszawa, Poland using apparatus under qualificationprocedure.


For statistical analysis Pearsonís correlationcoefficient has been applied.


Initially, the authors measured a decomposi-tion range of 3% DCP solution in propylene glycolwithout any additions. Results are shown in Table 1.The solution that was kept in a temperature of 4OCturned out to be stable, the degree of decompositionis negligible at least up to 14 days of observation(0.12%). This observation suggests that if ever DCPpreparations in propylene glycol would be availablecommercially they should be kept in a refrigeratorbecause sample stored at room temperature willdecompose slowly but visibly (2.38% after 14 days).

Further analysis of DCP samples with the addi-tion of 0.9% NaCl or water is shown in Tables 2-5

and Figure 1. The study included a series of meas-urements which ended after 70 h (except 3% DCPwith 1% of water). In both concentrations: 0.1% and3% with 1 : 1 proportions of 0.9% NaCl has beendecomposing during 70 h of observation. Itremained 86.4% and 81.1%, respectively, of itís ini-tial amount (Table 2 and 4). Correlation coefficientfor these data is statistically important measuring0.624. This means that 3% DCP decomposes quick-er than 0.1%. Meanwhile, the amount of diphenyl-acetylene (DPA), the product of DCPís decomposi-tion (0.1% DCP 1 : 1 0.9% NaCl) increased propor-tionally to DCPís decomposition (Table 2).Correlation coefficient for the DCP versus DPA lev-els (in 0.1% DCP solution) measures 0.989; hence.the more DCP disappears, the more DPA appears inlinear relationship, according to chemical equation,in which each molecule of DCP decomposes givingone molecule of DPA and one of carbon monoxide:DCP → DPA + CO (9). This proved accuracy ofprevious measurements of DCP. Above mentionedfindings suggest that large amounts of aqueous solu-tion accelerate decomposition of DCP though theydecomposes slower than the authors have expected.

Ultimately, the authors wanted to assign thetempo of DCP decomposition in a situation when asmall amount of water contaminated the sample,which may happen in any lab. The 1% (one masspercent) amount of water did not accelerate decom-position drastically; after 20 days there was still98.19% of initial DCP, which is below the error ofmeasurement (Table 5).

CONCLUSION

Very little is known about the way DCP reactswith human skin. It is hypothesized that at least

Figure 1. Graphical presentation of data from Tables 2-4


some molecules of DCP reach deep inside hair folli-cles since there was peribulbar and perivascularinfiltrate of lymphocytes after itís application (4, 8).Authors concluded that since human fluids containabout 0.9% NaCl (sweat a bit less) the addition of0.9% NaCl to DCP samples might be the first step toinvestigate itís stability in vivo. Of course humanskin is much more complex and it is to be expectedthat the stability of DCP in vivo would be dimin-ished by other factors: enzymes, keratin, immunesystem and others.

In fact, from a clinical point of view, it wouldbe best if the solutions of DCP, stable in a solventand in the refrigerator, would be less stable in vivo.The process of topical immunotherapy is to obtainmild allergic reaction which stops after a day or two,to avoid serious side effects. Paradoxically, anìunstableî allergen is advised here. From clinicalobservation (4), DCP might be exactly that kind ofallergen since serious allergic reactions to it aredescribed rarely. Moreover, most authors observedan allergic reaction which usually disappeared aftera day or two, therefore it is possible that there aremechanisms which inhibit or decompose DCP invivo. The results showed that after 70 h both sam-ples: with 0.1% DCP and 3% DCP still contain morethan 80% of primary DCP when mixed 1 : 1 with0.9% NaCl. It does explain the phenomenon of thedisappearing reaction to DCP only partially. Theremay be two scenarios to explain this phenomenon.The first one assumes that the product of DCPdecomposition ñ diphenylacetylene (DPA) acts asan inhibitor of DCP in vivo. That would be possiblesince many products of chemical reactions inhibitthe reaction itself. The second scenario suggests thatthere are other mechanisms in vivo, as mentionedabove, that decompose DCP or inhibit itís activity.

They include: the role of biological barriers, the roleof the immune system, reactions with proteins suchas keratin and others. These are numerous andrequire further investigation in the near future.

Acknowledgments

Authors would like to thank Dr. TomaszZieliÒski from National Defense University,Warsaw, for preparing statistical analysis and Edyta£πcka, M.Sc., Analytical Department, Institute ofIndustrial Organic Chemistry, Warsaw for chemicalanalysis.

REFERENCES

1. Alkhalifah A., Alsantali A., Wang E., McElweeK.J., Shapiro J.: J. Am. Acad. Dermatol. 62, 177(2010).

2. Damian D.L., Shannon K.F., Saw R.P.,Thompson J.F.: Australas. J. Dermatol. 50, 266(2009).

3. Gilhar A., Paus R., Kalish R.S.: J. Clin. Invest.117, 2019 (2007).

4. Herbst V., Zˆller M., Kissling S., Wenzel E.,Stutz N. et al.: Eur. J. Dermatol. 16, 537 (2006).

5. Hoffmann R., Wenzel E., Huth A., van derSteen P., Sch‰ufele M. et al.: J. Investig.Dermatol. 103, 530 (1994).

6. Hordinsky M., Ericson M.: J. Investig.Dermatol. Symp. Proc. 9, 73 (2004).

7. Kim Y.J.: Ann. Dermatol. 24, 373 (2012).8. Wasy≥yszyn T., Koz≥owski W., Zabielski S.L.:

Arch. Dermatol. Res. 299, 231 (2007).9. Wilkerson M.G., Henkin J., Wilkin J.K.: J. Am.

Acad. Dermatol. 11, 802 (1984).

Received: 19. 11. 2015


Pyrethroids are a class of synthetic insecticideswith chemical structure similar to that of naturalpyrethrum, but much more stable. They have beensubdivided into two classes based on their structuraldifferences, toxicological and neurophysiologicalactions. Structurally, type I pyrethroids (permethrinand tetramethrin) lack cyano substituent, whereastype II pyrethroids (fenpropathrin and deltamethrin)contain α-cyano group (1).

Pyrethroids have progressively replaced otherclasses of insecticides, such as organochlorines,organophosphates and carbamate derivatives, most-ly because of the notably high ratio between thetoxic dose in mammalian species and the effectivedose in target species (2). Moreover, pyrethroidshave become increasingly popular because of theirrelatively low costs, ease of application, and lengthof efficacy (usually lasting several weeks).Pyrethrins and pyrethroids are estimated at 23% ofthe insecticide world market, with more than 3500registered formulas. Pyrethroids, as well as tetram-ethrin, have been widely used in the field of agricul-ture, household (against flies, mosquitoes, cock-roaches, termites, and other harmful insects),forestry, horticulture and public health care (3).Tetramethrin has found its use in the treatment of

pediculosis in humans (4). Tetramethrin, among allsynthetic pyrethroids, is also extensively usedagainst a wide range of ectoparasites in large andsmall animals, with various insecticides used in dif-ferent formulations, including sprays, powders,lotions, shampoos, and aerosols (3).

There are numerous different instrumentaltechniques used for the determination of tetra-methrin. The available methods include voltammet-ric determination (5), capillary electrophoresis (6),spectrophotometric (7), as well as infrared spectro-metric methods (8). However, the most widelyapplied techniques are chromatographic methods,such as gas chromatography (GC) with electron cap-ture detection (ECD) (9) or with mass spectrometry(MS) (10) as well as high performance liquid chro-matography (HPLC) with diode array (DAD) (11) orwith MS (12) detection. The majority of analyticalmethods was developed for detection of trace levelsof the pesticides in environments like: air (13), soil(16), fruit, vegetable, water samples (14) or inhuman fluids (samples of breast milk, for example)(15). Chromatographic methods for tetramethrindetermination in diverse samples implicate some-what complex sample preparation, including solidphase extraction (12) or dispersive liquid microex-

OPTIMIZATION AND VALIDATION OF HPLC METHOD FOR TETRAMETHRIN DETERMINATION IN HUMAN SHAMPOO FORMULATION

MARINA Z. ZEKIC STOSIC*, SANDRA M. JAKSIC, IGOR M. STOJANOV, JELENA B. APIC and RADOMIR D. RATAJAC

Scientific Veterinary Institute ìNovi Sadî, RumenaËki put 20, 21000 Novi Sad, Serbia

Abstract: High-performance liquid chromatography (HPLC) method with diode array detection (DAD) wereoptimized and validated for separation and determination of tetramethrin in an antiparasitic human shampoo. Inorder to optimize separation conditions, two different columns, different column oven temperatures, as well asmobile phase composition and ratio, were tested. Best separation was achieved on the Supelcosil TM LC-18-DB column (4.6 ◊ 250 mm), particle size 5 µm, with mobile phase methanol : water (78 : 22, v/v) at a flow rateof 0.8 mL/min and at temperature of 30OC. The detection wavelength of the detector was set at 220 nm. Underthe optimum chromatographic conditions, standard calibration curve was measured with good linearity [r2 =0.9997]. Accuracy of the method defined as a mean recovery of tetramethrin from shampoo matrix was100.09%. The advantages of this method are that it can easily be used for the routine analysis of drug tetra-methrin in pharmaceutical formulas and in all pharmaceutical researches involving tetramethrin.

Keywords: HPLC, DAD, tetramethrin, determination, shampoo

1461

* Corresponding author: e-mail: [email protected]; phone: +381 64 818 54 79

1462 MARINA Z. ZEKIC STOSIC et al.

traction (14, 16). There are HPLC methods to deter-mine permethrin in different types of preparation(treated wood, lotion, creams, shampoo, etc.) (3, 17,18), but HPLC methods to qualify or identifytetramethrin as an active component in these prod-ucts are less known.

This paper, for the first time, presents anHPLC-DAD method of determination of tetra-methrin in shampoo against Pediculus capitis. Theaim of this study was to optimize and validate thedeveloped method of tetramethrin being an activecomponent, as well as to evaluate the suitability of anew method for quality control of tetramethrin con-tent in commercial shampoos.

EXPERIMENTAL

Chemicals and materials

The standard of tetramethrin (mixture of cis-and trans- isomers, 99.0%) was obtained fromEchrenstrofer GmbH, Germany. Organic solventsmethanol and acetonitrile (HPLC gradient grade)were obtained from Merck KGaA, Germany.Deionized water (WP 4100 reagent grade waterpurifier-SMEG) was used for standard and samplepreparations. SupitoxÆ shampoo, with declaredtetramethrin content of 0.3% against pediculosis,was purchased in a local market in Serbia. Matrixsolution that contains all substances present in theproduct that we analyzed (SupitoxÆ), but withoutactive component (tetramethrin) was obtained fromthe shampoo producer (Veterinary Institute,Subotica).

Chromatographic system

HPLC chromatographic apparatus consisted ofan HPLC Dionex UltiMate 3000 Series system witha DAD-3000SD/RS detector, autosampler WPS-3000(T)RS, degasser, binary pump HPG3200SD/RS and column oven TCC-3000SD(Thermo Scientific, Germany). The system control,data acquisition and data evaluation were performedby ChromeleonÆ7 software (Thermo Scientific,Germany).

Chromatography procedure

Mobile phase consisted of acetonitrile anddeionized water or methanol and deionized water,filtered through a 0.45 µm filter, degassed in anultrasonic bath, and pumped at a flow rate of 0.8mL/min. Injection volume was 20 µL. Comparedcolumns were Supelcosil TM LC-18-DB, 4.6 ◊ 250mm, 5 µm particle size (Sigma-Aldrich Co. LLC)and Hypersil GOLD aQ, 3 ◊ 150 mm, 3 µm particle

size (Thermo Scientific) at 30OC. The detectionwavelength of the detector was set at 220 nm for allused mobile phases.

Preparation of standard solution and shampoo

sample solution

The stock standard solution was prepared bydissolving 5 mg standard tetramethrin in 10 mL ofmethanol. The stock standard solution was kept inthe refrigerator at +4OC in an amber reagent bottle.For preparing the working standard solutions, 100,150, 200, 250, and 300 µL of the stock standardsolution were diluted to 10 mL in volumetric flaskwith mobile phase to obtain standard solutions inconcentrations 4.95, 7.425, 9.90, 12.375 and 14.85µg/mL of tetramethrin. Such prepared standard solu-tions were used for method development and valida-tion.

One milliliter of antiparasitic shampoo sample(SupitoxÆ) was accurately weighted into a 50 mLvolumetric flask and filled to the mark withmethanol. Then, working sample solution ofSupitoxÆ, was made by diluting 1.65 mL in 10 mLvolumetric flask with mobile phase to obtain 10 µgof tetramethrin per mL of sample solution. Thissample solution was filtered through a 0.45 µmPTFE filter to glass vial.

Twenty microliters of freshly prepared and fil-tered standard or sample solution was injected intothe HPLC system.


Since no method for determination of tetra-methrin in human antiparasitic shampoo with HPLC-DAD has been reported in literature, method devel-opment and optimization were done before valida-tion and sample analysis. Method parameters thatwere optimized are mobile and stationary phases.

Optimization of HPLC analysis and method

development

With the aim to develop fast and effective sep-aration of tetramethrin in short time, simple mobilephases were used for evaluation. In order to deter-mine the most suitable conditions for HPLC-DADseparation and detection of tetramethrin, two differ-ent columns and different mobile phase composi-tions in different ratios of organic and water phasewere used. Since acetonitrile provided lower viscos-ity, faster elution and better peak symmetry of insec-ticides comparing to methanol on reverse phase col-umn (3), mobile phase consisting of acetonitrile :water (55 : 45, v/v) was first chosen for evaluation.

Optimization and validation of HPLC method for tetramethrin determination... 1463

Figure 1. Separation of cis/trans- tetramethrin in matrix of antiparasitic shampoo; conditions: column 3 ◊ 150 mm, particle size 3 µm,mobile phase acetonitrile : water (55 : 45, v/v), flow rate 0.8 mL/min, temperature 60OC (A); column 3 ◊ 150 mm, particle size 3 µm,mobile phase methanol : water (78 : 22, v/v), flow rate of 0.8 mL/min, temperature 60OC (B); column 4.6 ◊ 250, 5 µm particle size, mobilephase methanol : water (78 : 22, v/v), flow rate of 0.8 mL/min, temperature 30OC (C)


In order to decrease mobile phase viscosity and tolower back-pressure in chromatography system (3),we used the column oven temperature of 60OC.Results showed that the mobile phase acetonitrile :water (55 : 45, v/v) on column Hypersil GOLD aQ(3 ◊ 150 mm, 3 µm), at a column oven temperatureof 60OC provides very poor separation of cis- andtrans- tetramethrin. As it can be seen from Figure1A, they have shown complete overlapping (peak2), while resolution (Rs) with the peak of matrix(peak 1) was 1.07. The maximum absorption peakfor tetramethrin was recorded at 220 nm, and that iswhy this wavelength was used to detect tetra-methrin.

According to literature data, methanol is a sol-vent that gives long retention time for permethrin(3). Because of that, to obtain acceptable resolutionfor tetramethrin isomers and sample matrix, acetoni-trile was replaced with methanol. Mobile phasemethanol : water (78 : 22, v/v) gave the completeseparation of cis- and trans- isomer peaks of tetram-ethrin (Fig. 1B). Still, there is a slight overlappingbetween tetramethrin and the peak of matrix solu-

tion (tR 4.203 min), since the resolution was unsatis-factory (Rs = 1.22) according to ICH guideline(2005). To provide better resolution of all peaks andcomplete separation, the column was replaced withthe longer one (4.6 ◊ 250 mm, 5 µm, Supelcosil TMLC-18-DB). Increasing the column length anddecreasing the column temperature to 30OC gave theacceptable peak base-separation with Rs values1.85. Optimal conditions for the separation oftetramethrin and matrix peaks established in thistrial were as follows: mobile phase that consisted ofmethanol : water (78 : 22, v/v), isocratic elution at aflow rate of 0.8 mL/min and temperature of 30OC.According to the requirements of ICH (2005), theseconditions were found to be most suitable for sepa-ration and quantification of both tetramethrin iso-mers concerning the resolution and peak symmetry,with relatively short time of analysis (Fig. 1C).

Validation of the developed method

In order to evaluate reliability of the results,validation of the method included the evaluation ofperformance parameters such as specificity, selec-

Figure 2. Chromatograms of matrix solution (A) and separation of cis/trans- tetramethrin in standard solution (B) and antiparasitic sham-poo sample (C)

Table 1. HPLC system suitability parameters for tetramethrin isomers determination.

Cis-tetramethrin Trans-tetramethrin

Retention time (min) 8.09 8.73

Retention time repeatability RSD (%)a 0.38 0.27

Peak symmetry 0.97 1.12

Resolution 2.21 1.76

a Made in six replicates

Optimization and validation of HPLC method for tetramethrin determination... 1465

tivity, linearity, precision and accuracy. Within themethod validation, the parameters of the HPLC sys-tem suitability test (SST) were also measured andevaluated. Mean values and standard deviations ofretention time, peak asymmetry, resolution, andrepeatability of analytical run were calculated fromthe results of six standard solution injections(Table1).

Specificity and selectivityFor chromatographic procedures, representa-

tive chromatograms should be used to demonstratespecificity and individual components should beappropriately labeled (19). For the purpose of deter-mining the specificity and selectivity of the testedanalytical method the obtained chromatograms formatrix, standard solution and the solution of theexamined shampoo sample were compared (Fig. 2).As can be seen from this Figure, chromatograms areshowing that there is no peak overlapping. Othersubstances potentially present in the shampoo,besides the main active ingredient, are of no influ-ence on the analysis, thus, precise determination ofactive ingredient is possible even in the presence ofmatrix interferences.

LinearityLinearity study was performed by testing five

test solutions at concentration levels from 50 to150% of the target analyte concentration, in therange 4.95ñ14.85 µg/mL. Each concentration of cal-ibration standard was measured in triplicates. Underthe tested chromatographic conditions, linear rela-tionships of standard solutions were verified for thesum of both cis- and trans- tetramethrin. The detec-tor response was linear in the whole range of the cal-ibration curve. The linearity was evaluated by linearregression analysis, which was calculated by theleast square regression method. The peak area wasplotted against tetramethrin concentration at eachlevel. By linear regression analysis, good linearity [r = 0.99985] was achieved in the researched range.Linear regression parameters were described by the

following equation: A = 1.5079c + 0.462 for totalcis- and trans- tetramethrin.

PrecisionIn order to determine the precision of the

method, standard solution containing 9.90 µg/mL oftetramethrin standard was analyzed. A number often measurements were analyzed in one day.Precision was expressed as relative standard devia-tion (RSD) value, which was 0.26%. Since thisresult showed a value less than 2%, the intraday pre-cision of the method is in accordance with ICHguidelines.

Accuracy The accuracy of the method was evaluated by

determination of tetramethrin in spiked sampleswithin the same day. Spiked samples were preparedby spiking of the sample matrix with different con-tent of tetramethrin standard solution (added amount80-120% of tetramethrin content) in triplicates. Theaccuracy was calculated as deviation of the meanfrom the nominal concentration within the day(spike recovery). As it can be seen from the resultsshown in Table 2, recovery value is in an acceptablerange of 100 ± 2% (19).

Tetramethrin determination in shampoo sample

An optimized and validated method has beenapplied to determination of tetramethrin in antipara-sitic shampoo SupitoxÆ. Shampoo sample wascommercially available in the local market. Theestablished average amount (3 determinations) oftetramethrin was 0.305% of cis/trans-tetramethrin.

CONCLUSION

In this paper, we developed and validated a fastand simple HPLC-DAD method for separation ofcis/trans- tetramethrin and determination of totaltetramethrin in antiparasitic shampoos. All valida-tion parameters were within an acceptable rangeaccording to ICH (2005). The developed method

Table 2. Accuracy of tetramethrin determination by optimized method.

Concentration of spiked samples(µg/mL)*

% of target concentration Recovery ± SD (%)

7.92 80 99.71+0.62

9.90 100 99.43+0.71

11.88 120 101.13+0.85

*Made in three replicates


was successfully applied to estimate the amount oftetramethrin in the shampoo against pediculosis.This method can be used for the routine analysis.

Acknowledgment

This work was supported by the scientific proj-ect grant TR 031071 of Ministry of Education andScience of the Republic of Serbia


The authors declare that they have no conflictof interests.

REFERENCES

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10. Feo M.L., Eljarrat E., BarcelÛ D.: J.Chromatogr. A 1217, 2248 (2010).

11. Loper B.L., Anderson K.A.: JAOAC Int. 86,1236 (2003).

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13. Raeppel C., Appenzeller B.M., Millet M.:Talanta 131, 309 (2015).

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19. ICH VALIDATION OF ANALYTICAL PRO-CEDURES: TEXT AND METHODOLOGYQ2(R1), ICH Harmonized Tripartite Guideline,International Conference on Harmonization ofTechnical Requirements for Registration ofPharmaceuticals for Human Use. Geneva 2005.

Received: 19. 11. 2015


There has been a significant increase in variousallergic disorders over the last few decades. Theessential medications used for the treatment of aller-gies are antihistamines.

The mechanism of action of antihistamines isbased on their competitive and reversible binding tothe H1 receptor that results in the abolition of theeffects of histamine as a possible chemical mediatorimplicated in inflammation and allergic reactions.

In general, two groups of antihistamines can bedistinguished: first generation and second genera-tion types.

First generation H1-antihistamines have poorH1 receptor selectivity, and thus they may also blockother receptors causing a number of side effectssuch as dryness of mucous membranes, arrhythmia,urination disorders, tachycardia, dizziness,increased appetite. In addition, they have the abilityto cross the blood-brain barrier by inhibiting thephysiological effects of histamine, and leading todrowsiness, visual disturbances, memory deficitsand cognitive disorders.

In modern medicine, second-generation anti-histamines play a fundamental role in the treatmentof allergic disorders. Second generation H1-antihist-

amines are highly selective for the histamine H1

receptor and do not cross the blood-brain barrier (orcross only to a small extent). Second generationantihistamines differ from first generation becauseof their high selectivity and the lack of effect on thecentral nervous system (CNS). Therefore, secondgeneration antihistamines exerted much less sideeffects compared with first generation antihista-mines.

Modern second generation antihistamines(which are often metabolites or isomers of olderdrugs such as desloratadine ñ a product of loratadinemetabolic transformation, or fexofenadine ñ a prod-uct of terfenadine metabolic transformation) alsoexhibit non-receptor anti-inflammatory effects (e.g.,desloratadine and rupatadine) (1).

Rupatadine (Fig. 1) is a novel antihistamine ofthe second generation that was introduced intoEurope for the treatment of allergic disorders. Itsproperties go beyond the characteristics of otherdrugs that belong to the group of second generationantihistamines. One moiety of this molecule hashigh affinity to H1 receptor, and the second oneblocks the receptor for PAF (platelet activating fac-tor). Rupatadine is able to work against both early-

IDENTIFICATION AND DETERMINATION OF RUPATADINE AND FEXOFENADINE BY DENSITOMETRIC METHOD

KRYSTYNA CZERWI—SKA1*, ELØBIETA WYSZOMIRSKA1, ELØBIETA KUBLIN1, EWA MALANOWICZ1 and ALEKSANDER P. MAZUREK1,2

1Department of Basic and Applied Pharmacy, National Medicines Institute,30/34 Che≥mska St., 00-725 Warszawa, Poland

2Department of Drug Chemistry, Medical University of Warsaw, 1 Banacha St., 02-097 Warszawa, Poland

Abstract: Simple, precise and accurate densitometric methods were developed for the determination of twoantihistamine drugs, rupatadine and fexofenadine. Silica gel 60 F254 HPTLC plates were used as stationaryphase, while mixtures of acetonitrile ñ water ñ 25% ammonia (90 : 10 : 1, v/v/v) and acetonitrile ñ methanol ñacetate buffer at pH 5.5 (3 : 2 : 5, v/v/v) were used as mobile phases for rupatadine and fexofenadine, respec-tively. The detection of rupatadine and fexofenadine was conducted out at 256 and 210 nm, respectively. Thelimit of detection and the limit of quantification for rupatadine were found to be 0.3 and 0.1 µg/spot, respec-tively, and for fexofenadine, 5 and 2 µg/spot, respectively.

Keywords: rupatadine fumarate, fexofenadine hydrochloride, antihistamine drugs, densitometric method

1467


1468 KRYSTYNA CZERWI—SKA et al.

and late-stage allergic reactions. In the early stage -through the antihistamine activity, in the later -through both the anti-inflammatory and anti-PAFeffects.

Rupatadine is safe, not cardiotoxic and doesnot impair psychomotor or cognitive activity. Thedrug is well tolerated, with mild and moderate sideeffects. Rupatadine is highly effective in the treat-ment of allergic rhinitis, conjunctivitis and urticaria(1).

Fexofenadine (Fig. 2) is a modern secondgeneration antihistamine. This antihistaminic drugpotently and selectively blocks histamine H1 recep-tors, thus it has strong anti-allergic properties.Fexofenadine penetrates into CNS to a smallextent, and does not cause sedation and drowsinessas side effects. It is used for the symptomatic treat-ment of allergic rhinitis and chronic idiopathicurticaria (2).

A review of the literature of the last five yearsreveals that rupatadine was determined in tabletswith the following methods: spectrophotometricmethod at 244 nm (3), spectrophotometric method

after conversion into rupatadine derivatives at 416,511 and 527 nm (4) and HPLC method with UVdetection on column C18 with a mixture ofmethanol ñ sodium phosphate monobasic ñ acetoni-trile as mobile phase (5).

Fexofenadine was determined by a spectropho-tometric method after conversion to fexofenadinederivatives at 612 and 615 nm (6), spectrophotomet-ric method using the first derivative (7), spectroflu-orometric method (fluorescence intensity was meas-ured at 612 and 615 nm (8), HPLC method per-formed on a Zorbax Eclipse XDB-C8 (9, 10) col-umn with a mixture of ammonium acetate ñ formicacid ñ methanol (1 mmol/L) as mobile phase andMS detector (9), or with a mixture of phosphatebuffer ñ acetonitrile ñ methanol (1 mmol/L) asmobile phase and UV detector (10), and UPLCmethod, in gradient (with the following mobilephases: 0.1% formic acid and 1% formic acid withacetonitrile) performed on a column C18 with UVdetection (11). Fexofenadine was determined inmedicinal products (6-8, 10) and in plasma (9, 11).

Antihistamines are currently a widely usedgroup of drugs. Therefore, an attempt was made todevelop simple and inexpensive methods of theirdetermination.

Several methods for the identification (TLC)and densitometric methods for the determination ofantihistamines: loratadine, clemastine, prometh-azine, ketotifen, cetirizine, desloratadine, dimetin-dene and azelastine (12, 13) were developed in pastyears at the Department of Basic and AppliedPharmacy of National Medicines Institute.

The aim of this study was to develop a sensi-tive, simple and cost-effective methods for thedetermination of two modern antihistaminic drugs,rupatadine and fexofenadine.

Figure 1. 8-Chloro-6,11-dihydro-11-{1-[(5-methyl-3-pyridinyl)methyl]-4-piperidinylidene}-5H-benzo[5,6]cyclohepta[1,2-b]pyridine(rupatadine)

Figure 2. 4-{Hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidin-yl]butyl}-α,α- dimethylbenzeneacetic acid (fexofenadine)

Identification and determination of rupatadine and fexofenadine by... 1469

EXPERIMENTAL

Reagents and equipment

ñ analytically pure and high purity reagents forHPLC by Rathburn;

ñ chromatographic plates: HPTLC silica gelF254 and HPTLC silica gel RP18 F254s by Merck;

ñ Hanau UV lamp;ñ Camag Automatic TLC sampler;ñ Shimadzu CS-9000 scanning densitometer;ñ liquid chromatograph Dionex Ultimate 3000

with a UV detector.

Materials

Reference materials:● Rupatadine fumarate Ref. St. batch RUPAF-0910● Fexofenadine hydrochloride Ref. St. batch 1.0Tested substances:● Rupafin, 10 mg tablets (Rupatadine fumarate),

J.Uriach & Cia, S.A.● Telfast 120, 120 mg film-coated tablets

(Fexofenadine hydrochloride), Aventis

QUALITATIVE ANALYSIS

Identification of rupatadine fumarate and fexofe-nadine

At the first stage of the studies, optimal chro-matographic systems were investigated for the iden-tification of examined substances, rupatadine andfexofenadine.

Methanolic standard solutions of rupatadinefumarate and fexofenadine were prepared at the fol-lowing concentrations: 1, 0.1 and 0.01 mg/mL.

The following amounts: 30, 20, 10, 5, 2.5, 2,1.5, 1, 0.5, 0.25, 0.2, 0.1, 0.05 and 0.025 µg of

Figure 3. UV spectrum of rupatadine measured directly from the TLC plate

Figure 4. Densitogram of rupatadine at 256 nm


examined substances were spotted on Merck silicagel F254 HPTLC or Merck silica gel RP 18 F254S

HPTLC plates. The chromatograms were thendeveloped with various mobile phases. After airdrying, the positions of the spots were examinedunder UV light at 254 nm and visualized in iodinevapor.

Several mobile phases for the qualitativeanalysis have been examined and the followingseven HPTLC systems were selected:I. Merck silica gel F254 HPTLC plates and mobilephase: chloroform ñ methanol ñ 25% ammonia (90 :10 : 1, v/v/v), with chamber saturation;II. Merck silica gel F254 HPTLC plates and mobilephase: diethyl ether ñ diethylamine (40 : 1, v/v);III. Merck silica gel F254 HPTLC plates and mobilephase: methanol - 25% ammonia (100 : 1.5, v/v);IV. Merck silica gel RP 18 F254S HPTLC plates andmobile phase: acetonitrile ñ methanol ñ acetatebuffer at pH 5.5 (3 : 2 : 5, v/v/v);V. Merck silica gel F254 HPTLC plates and mobilephase: toluene ñ ethyl acetate ñ acetic acid (16 : 4 :1, v/v/v);VI. Merck silica gel F254 HPTLC plates and mobilephase: n-heksane ñ acetone ñ 25% ammonia (85 : 14: 1, v/v/v);VII. Merck silica gel F254 HPTLC plates and mobilephase: acetonitryl ñ water ñ 25% ammonia (90 : 10: 1, v/v/v).

In qualitative analysis, the Rf values were cal-culated and the limits of detection of examined com-pounds were determined.

Obtained results are given in Table 1.

QUANTITATIVE ANALYSIS

Determination of rupatadine fumarateThe densitometric method was used for the

determination of rupatadine fumarate.Two systems were selected for further test-

ing. System VII (described in section consideringidentification of rupatadine): Merck silica gel F254

HPTLC plates and mobile phase: acetonitrile ñwater ñ 25% ammonia (90 : 10 : 1, v/v/v) wasselected for the quantitative determination ofrupatadine, and system IV (described in sectionconsidering identification of fexofenadine): Mercksilica gel F254 HPTLC plates and mobile phase:acetonitrile ñ methanol ñ acetate buffer at pH 5.5(3 : 2 : 5, v/v/v) was selected for the determinationof fexofenadine. The maximum wavelengthsdetermined were 256 nm and 210 nm for rupata-dine and fexofenadine, respectively (Fig. 1 andFig. 3).

Tab

le 1

. Rf

valu

es a

nd li

mits

of

dete

ctio

n fo

r th

e te

sted

com

poun

ds (

Gra

y Fi

elds

ref

er to

the

mob

ile c

hase

sel

ecte

d fo

r qu

antit

ativ

e de

term

inat

ion

of a

giv

en c

ompo

und.

Syst

em I

Syst

em I

ISy

stem

III

Syst

em I

VSy

stem

VSy

stem

VI

Syst

em V

II

Tes

ted

Lim

it of

L

imit

of

Lim

it of

L

imit

of

Lim

it of

L

imit

of

Lim

it of

co

mpo

und

Rf

dete

ctio

n [µ

g]R

fde

tect

ion

[µg]

Rf

dete

ctio

n [µ

g]R

fde

tect

ion

[µg]

Rf

dete

ctio

n [µ

g]R

fde

tect

ion

[µg]

Rf

dete

ctio

n [µ

g]

UV

iodi

neU

Vio

dine

UV

iodi

neU

Vio

dine

UV

iodi

neU

Vio

dine

UV

iodi

ne

Rup

atad

ine

0.91

0.25

0.25

0.28

0.1

0.25

0.85

0.25

0.25

0.0

0.1

-0.

00.

250.

250.

00.

250.

50.

320.

10.

25

Fexo

fena

dine

010

10

1010

0.95

51

0.36

21

010

0.5

010

0.2

0.24

100.

2

Chr

omat

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sys

tem

s nu

mer

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n -

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text

.


All densitometric measurements were donewith the Shimadzu CS 9000 densitometer.Chromatographic plates were placed within the den-sitometer chamber. The slit dimension was kept at0.4 ◊ 0.4 mm. Densitometric scanning was per-former in a zigzag mode, while the width of deflec-tion was 10 and 16 mm for rupatadine and fexofe-nadine, respectively (Fig. 2 and Fig. 4).

Determination of regression curvesIn order to perform a regression analysis the

solutions of standards and tablet samples were pre-pared.

The standard solutions of rupatadine fumarateand sample solutions obtained from Rupafin 10 mgtablets of 0.05, 0.075, 0.1, 0.15, 0.2 and 0.3 mg/mLwere prepared in methanol.

The standard solutions of fexofenadine andsample solutions obtained from Telfast 120 mgtablets of 0.5, 1.0, 2.5, 4, 5 and 6 mg/mL were pre-pared in methanol.

The samples were shaken for 14 min into anultrasonic bath, then, they were shaken for 30 min ina mechanical shaker. The solutions were filteredthrough filters with a pore size of 0.45 µm.

The 10 µL portions of standards and tabletsamples solutions were spotted on the plates.

The developed chromatograms were air driedand visualized under UV light at 254 nm. Then, den-sitometric measurements were made with wave-lengths determined prior to analysis.

The regression curves were plotted. For theanalyzed compounds linearity was proven; forrupatadine in the range of 0.5-3 µg (Fig. 5) and forfexofenadine in the range of 5-60 µg (Fig. 6).

Determination of content in medical products● Rupafin ñ 10 mg tablets (Rupatadine fumarate)● Telfast 120 ñ 120 mg film-coated tablets

(Fexofenadine hydrochloride)The sample solutions of rupatadine (0.15 mg/mL)

and fexofenadine (1 mg/mL) were prepared from com-mercially available Rupafin 10 mg tablets and Telfast120 mg film coated tablets, respectively. The corre-sponding standard solutions for both examined sub-stances were also prepared at the same concentrations.

The isolation of components from tablets wasconducted by means of methanol. Samples wereshaken for 14 min in an ultrasonic bath, then, theywere shaken for 30 min in a mechanical shaker.

Table 2. Statistical assessment of the results concerning the determination of the tested compounds in pharmaceutical products.

Tested Number of Arithmetic mean of Standard Confidence interval RSD compound Method samples all measurments deviation S X ± ∆X [%]

X [mg] PU = 95%

Rupafin tablets Densitometry 10 9.950 mg 0.04 9.950 ± 0.057 mg 0.4110 mg

(Rupatadine fumarate) HPLC 10 9.910 mg 0.13 9.910 ± 0.079 mg 1.38

Telfast 120 coated Densitometry 7 120.65 mg 0.73 120.65 ± 0.79 mg 0.60tablets 120 mg(Fexofenadine) HPLC 7 120.46 mg 0.70 120.46 ± 0.75 mg 0.58

Figure 5. UV spectrum of fexofenadine mesured directly from the TLC plate


Solutions were filtered through filters with a 0.45µm pore size. The 10 µL portions of standard andsample solutions were transferred on silica gel F254

HPTLC plates. After development with the mobilephase appropriate for each compound, the chro-matograms were dried and the densitometric analy-sis was performed at determined wavelengths.

The results and statistical analysis are shown inTable 2.

In order to compare developed methods for thedetermination of examined substances in medicinalproducts, comparative analysis of rupatadine and fexo-fenadine was performed by HPLC methods describedin the literature (7, 11). Fexofenadine was determinedon a Zorbax C-8 (5 µm, 4.6 mm ◊ 150 mm) columnwith UV detector at 210 nm. The mobile phase con-sisted of buffer acetate ñ acetonitrile ñ methanol in theratio 60 : 20 : 20, v/v/v adjusted to pH 3.7 (7).Rupatadine was determined on a column LichrosphereC-8 (5 µm, 4.6 mm ◊ 250 mm) with UV detector at226 nm. The following mobile phase was used:methanol ñ buffer solution of potassium dihydrogenphosphate ñ acetonitrile (50 : 20 : 30, v/v/v) (11).

The obtained results are presented in Table 2.

RESULTS

Several different mobile phases and differenttypes of chromatographic plates were investigated. Forthe qualitative analysis of both examined substancesseven chromatographic system were selected. Rf val-ues and detection limits are summarized in Table 1;systems are described in section about identification.

Obtained results show that systems II, III andVII are the best to confirm the identity of rupata-

Figure 7. Regression curve (dependence of chromatogram peak area (y) from the amount of rupatadine fumarate (x).

Figure 6. Densitogram of fexofenadine at 210 nm


dine fumarate. System IV and optionally V or VIcan be used to establish the purity of the compound.For best results, system IV and optionally III or VIIare used to confirm the identity of fexofenadine.Other systems (I, II, V and VI) may be used toassess purity of the compound. Rf values andbroadening/blurr of chromatographic spots weretaken into consideration when selecting system forquantitative analysis.

The following systems were used for quantita-tive analysis. System VII: Merck silica gel F254

HPTLC plates and mobile phase: acetonitrile ñ waterñ 25% ammonia (90 : 10 : 1, v/v/v) was selected forthe determination of rupatadine (Rf value of ca. 0.32).

System IV: Merck Silica gel RP 18 F254S

HPTLC plates and mobile phase: acetonitrile ñmethanol ñ acetate buffer at pH 5.5 (3 : 2 : 5, v/v/v)proved to be the best for the quantification of fexo-fenadine (Rf value of ca. 0.36).

Automatic sample applications were used toensure the highest precision of densitometric meas-urements.

Conformity of chromatograms of examined sub-stances isolated from Rupafin 10 mg tablets andTelfast 120 mg coated tablets and standard substanceswas demonstrated for all analyzed compounds.

Linearity was shown for both analyzed com-pounds in the range of 0.5-3.0 mg for and 5-60 mgfor rupatadine and fexofenadine, respectively.

Coefficients of determination R2 were 0.999and 0.9985 for rupatadine and fexofenadine, respec-tively. Equations of curves are shown in Figures 5,6. The limits of quantification were 0.3 µg and 5 µgfor rupatadine and fexofenadine, respectively. Thelimits of detections were 0.1 µg and 2 µg for rupata-dine and fexofenadine, respectively.

Simultaneously, in order to compare developedmethods for the determination of examined sub-

stances in medicinal products, comparative analysisof discussed compounds was performed by HPLCmethods described in the literature. The results aregiven in Table 2.

The statistical data obtained for developed den-sitometric methods and for comparative methods aresimilar.

CONCLUSIONS

Developed chromatographic system: Mercksilica gel F254 HPTLC plates and mobile phase: ace-tonitrile ñ water ñ 25% ammonia at (90 : 10 : 1,v/v/v) ensures optimal identification conditions forrupatadine fumarate in Rupafin tablets 100 mg.

Developed chromatographic system: Mercksilica gel F254 HPTLC plates and mobile phase: ace-tonitril ñ methanol ñ acetate buffer at 5.5 (3 : 2 : 5,v/v/v) ensures optimal identification conditions forfexofenadine in Telfast tablets 120 mg.

Developed systems were used in densitometricdetermination of rupatadine fumarate and fexofenadine.Statistical data obtained for the developed assay meth-ods indicate their sufficient precision and accuracy.

Not only are developed densitometric methodscomparable to HPLC in terms of accuracy and pre-cision, but also are cheaper and quicker to conduct.Hence, they can be routinely used to determinaterupatadine fumarate and fexofenadine in medicalproducts.

REFERENCES

1. Grzelewska-Rzymowska I., GÛrski P.: Post.Dermatol. Alergol. 6, 489 (2011).

2. Charakterystyka Produktu Leczniczego Telfast120, 120 mg tabletki powlekane firmy SanofiAventis.

Figure 8. Regression curve (dependence of chromatogram peak area (y) from the amount of fexofenadine hydrochloride (x).


3. Choudekar R.L., Mahajan M.P., Sawant S.D.:Int. J. Pharm. Pharm. Sci. 4, Suppl. 3, 737(2012).

4. Rele R.V., Patil S.S.: Der Pharma Chemica 4,2278 (2012).

5. Gandla K., Spandana R., Kumar J.M.R.,Surekha M.L., Sheshgiri Rao J.V.L.N.: DerPharma Chemica 4, 1819 (2012).

6. Narayana B., Veena K.: Indian J. Chem. Techn.17, 386 (2010).

7. Tavallali H., Nori K.: Int. J. Chem. Tech. Res.5, 2471 (2013).

8. Abd El-Hay S.S., Colyer C.L., Hassan W.S.,Shalaby A.: J. Fluoresc. 22, 247 (2012).

9. Chen Y., Chou H., Chang W., Hsu K.: J.Chinese Pharm. Sci. 22, 409 (2013).

10. Nimje H.M., Nimje S.T., Oswal R.J., BhamreS.T.: e-J. Chem. 9, 1257 (2012).

11. Tanaka Y., Yoshikawa Y., Yasui H.: Biol.Pharm. Bull. 35, 698 (2012).

12. CzerwiÒska K., Wyszomirska E., MazurekA.P.: Acta Pol. Pharm. Drug Res. 70, 19 (2013).

13. Wyszomirska E., CzerwiÒska K., Kublin E.,Mazurek A.P.: Acta Pol. Pharm. Drug Res. 70,951 (2013).

Received: 25. 11. 2015


Hyperlipidemia and hypertension are theimportant risk factors for atheroma. Atheromatousdisease is ubiquitous and underlies the commonestcause of death (myocardial infarction caused bythrombosis). High plasma LDL cholesterol is a riskfactor of cardiovascular diseases, such as athero-sclerosis, which is characterized by deposition ofcholesterol on the arterial wall. Atherosclerosis ofthe coronary and peripheral vasculature is the lead-ing cause of death worldwide. When change in thelifestyle and in the diet is not enough, a medicaltreatment is necessary. In the therapy of these dis-eases combination of antihypertensive, hypolipi-demic and antidiabetic drugs is often used.

Acidic drugs

Statins are commonly used to treat severalforms of hypercholesterolemia in primary and sec-ondary prevention, they possess high effectiveness inreducing total cholesterol and low-density lipopro-tein cholesterol levels in human body. Atorvastatin ismost frequently prescribed in combined formulationswith different drugs. However, statins exhibit a num-

ber of adverse effects, such as myopathy or rhab-domyolysis, so it is necessary to monitor the levels ofstatins in order to establish an appropriate dosagescheme, which would minimize adverse effects andkeep the cholesterol-lowering effect.

Analysis of acidic drugs

To date, several HPLC-UV (1-6) and HPLC-MS (7, 8) methods have been developed for thequantification of these drugs either alone or in com-bination with other drugs in different matrices.

Several chromatographic methods have beenreported for the qualitative and quantitative determi-nation of statins in biological samples e.g., humanserum (3), human plasma (10, 11), wastewater sam-ples (4, 7) and pharmaceutical formulations (1, 2, 5,6, 12-15).

β-Blockers were also determined in differentsamples e.g., whole blood (16), human plasma (17,18), urine (19) and pharmaceutical formulations(20).

Analysis of acidic compounds was often per-formed with mobile phases containing addition of

RP-HPLC ANALYSIS OF ACIDIC AND BASIC DRUGS IN SYSTEMS WITHDIETHYLAMINE AS ELUENTS ADDITIVE

ANNA PETRUCZYNIK*, KAROL WR”BLEWSKI, SZYMON STR”ØEK and MONIKA WAKSMUNDZKA-HAJNOS

Department of Inorganic Chemistry, Medical University of Lublin, Chodüki 4a, 20-093 Lublin, Poland

Abstract: The chromatographic behavior of some basic and acidic drugs was studied on C18, Phenyl-Hexyland Polar RP columns with methanol or acetonitrile as organic modifiers of aqueous mobile phases containingaddition of diethylamine. Diethylamine plays a double function of silanol blocker reagent in analysis of basicdrugs and ion-pair reagent in analysis of acidic drugs. Most symmetrical peaks and highest system efficiencywere obtained on Phenyl-Hexyl and Polar RP columns in tested mobile phase systems compared to resultsobtained on C18 column. A new rapid, simple, specific and accurate reverse phase liquid chromatographicmethod was developed for the simultaneous determination of atorvastatin - antihyperlipidemic drug andamlodipine - calcium channel blocker in one pharmaceutical formulation. Atorvastatin is an acidic compoundswhile amlodipine is a basic substance. The chromatographic separation was carried out on Phenyl-Hexyl col-umn by gradient elution mode with acetonitrile as organic modifier, acetate buffer at pH 3.5 and 0.025 M/Ldiethylamine. The proposed method was validated for specificity, precision, accuracy, linearity, and robustness.The linearity range of atorvastatin and amlodipine for 5 ñ 100 µg/mL was obtained with limits of detection(LOD) 3.2750 µg/mL and 3.2102 µg/mL, respectively. The proposed method made use of DAD as a tool forpeak identity and purity confirmation.

Keywords: acidic and basic drugs, RP-HPLC, silanol blocker, ion-pair chromatography

1475


1476 ANNA PETRUCZYNIK et al.

acids or buffer at acidic pH for suppression of ana-lytes ionization. Rarely, acidic analytes was deter-mined in systems containing addition of ion pairreagents.

Different chromatographic systems wereapplied for determination of investigated drugs,but most analyses were performed on C18 orrarely C8 columns with mobile phases containingorganic modifier (methanol or acetonitrile), waterand addition of acids e.g., trifluoroacetic acid (3),formic acid (9, 23), phosphoric acid (5, 21, 22) orbuffers at acidic pH (1, 8, 10, 14, 24-26),. Rarely,determination of these drugs was performed onCN (13) or phenyl (2) columns. Application ofmobile phases with addition of different ion pairreagents were also reported e.g., timolol, rosuvas-tatin and diclofenac were analyzed in chromato-graphic system containing mobile phase with addi-tion of triethylamine (11). Triethylamine was alsoadded to mobile phase for determintion of telmi-sartan in bulk drugs and formulations on C18 col-umn (13).

Basic drugs

Telmisartan, valsartan, losartan are theangiotensin II type receptor antagonist blockers,

widely used in treatment of hypertension.Benazepril and cilazapril are potent angiotensinconverting enzyme inhibitors and they are used inthe treatment of hypertension and congestive heartfailure.

β-Blockers are widely used as standard thera-peutics in the treatment of high blood pressure,arrhythmias, and angina pectoris. Besides, β-block-ers also improved the heartís ability to relax andexhibit calming neurological effects decreasing anx-iety and stabilizing motor performance.

Carvedilol is an antagonist of α1 and β1, β2membrane adrenoreceptors and also a modulator ofcardiac electrophysiological properties.

Alkaloid ephedrine is a sympathomimetic ago-nist at both α- and β-adrenergic receptors, whichdetermines an increase of cardiac rate and contrac-tility, peripheral vasoconstriction, bronchodilatationand central nervous system (CNS) stimulation.Caffeine acts as a CNS stimulant.

Amlodipine is a dihydropyridine derivativewith calcium antagonist activity. It is used in themanagement of hypertension, chronic stable anginapectoris and prinzmetal variant angina. Amlodipineinhibits the transmembrane influx of calcium ionsinto vascular smooth muscle and cardiac muscle.

Table 2. Values of tR, AS, and N/m for investigated acidic drugs obtained on different columns in eluent system containing MeOH, acetatebuffer at pH 3.5 and 0.025 M/L DEA.

C18 Phenyl-Hexyl Polar RP

Name of60% MeCN, acetate 60% MeOH, acetate 50% MeOH, acetate

drugbuffer at pH 3.5 and buffer at pH 3.5 and buffer at pH 3.5 and

0.025 M/L DEA 0.025 M/L DEA 0.025 M/L DEA

tR AS N/m tR AS N/m tR AS N/m

Atorvastatin 9.03 0.77 7294 18.45 1.04 17766 24.61 1.03 18980

Fluvastatin 12.31 0.93 15277 25.81 1.16 29843 21.25 1.08 25390

Rosuvastatin 2.94 0.70 4027 6.42 0.88 9802 9.02 0.88 13502

Pravastatin 2.65 0.63 2730 4.14 0.74 5222 4.64 0.71 6221

Valsartan 3.32 0.64 2330 5.43 0.77 5118 7.45 1.14 3499

Telmisartan 11.3 0.73 10577 23.89 1.09 24556 71.08 1.34 21291

Benazapril 3.83 0.73 5395 6.67 0.95 11514 9.31 1.01 14902

Cilazapril 3.39 1.14 10901 5.92 1.08 18120 8.8 1.10 23303

Table 1. Gradient elution program.

Eluent A Eluent B Eluent CTime H20 ACN Acetate buffer

+ 0.025 M/L + 0.025 M/L + 0.025 M/L DEA (% v/v) DEA (% v/v) DEA (% v/v)

0 40 40 20

10 25 55 20

RP-HPLC analysis of acidic and basic drugs in systems with... 1477

Analysis of basic drugs

Basic compounds can interact with underiva-tized free silanol groups of silica-based chemicallybonded stationary phases. This situation leads topeak tailing, increased retention, and low systemefficiency. Interactions with the silanols can bereduced by application of mobile phases at low pH,when silanol ionization is suppressed, or at high pH,to suppress solute ionization. In analysis of basiccompounds anionic ion-pair reagents are rarely usedto form neutral associates. Good results for analysisof basic compounds is achieved by use of mobilephases containing organic amines as silanol block-ers.

Basic drugs such as β-blockers in most caseswere also analyzed on C18 columns with mobilephases containing organic modifier and addition ofacids (27, 28), buffer at acidic pH (16, 20, 29, 30),buffer at basic pH (18), amines e.g., triethylamine(19). Chromatographic systems with mobile phasescontaining ion-pair reagents were also applied fordetermination of these drugs. Zarghi et al. analyzedsotalol in plasma samples on C18 column with mix-ture of acetonitrile, sodium dihydrogen phosphateand heptane sulfonic acid (18).

Carvedilol was analyzed in tablets on C18 col-umn with mixture of acetonitrile and phosphatebuffer at pH 2.5 as eluent (31), or in human plasmaon CN column with mobile phase containing addi-tion of triethylamine (32). Ephedrine was deter-mined on PFP column with mobile phase containingacetonitrile, water and ammonium acetate (33).Addition of ion-pair reagents: 1-octanesulfonic acidsodium salt and tetrabutylammonium hydrogen sul-fate was also applied for determination of nicergo-line (34).

Torasemide, belonging to the class of loopdiuretics with a high ceiling effect, was determinedon CN column with a mobile phase containing ace-tonitrile and aqueous solution of perchloric acid(35).

Antihypertensive basic drugs were often ana-lyzed on C18 column with mobile phases containingaddition of acids (36-39).

Amines were also added to mobile phases foranalysis of basic drugs for blocking free silanolgroups on the surface of stationary phases.Amlodipine was determined in eluent systems con-taining triethylamine (40, 41).

Different drugs from these groups were simul-taneously analyzed by HPLC. Determination ofolmesartan, medoxomile, amlodipine and hydro-chlorothiazide in tablet dosage form was performedon C18 column with mobile phase containing ace-

tonitrile, aqueous solution of phosphoric acid andtriethylamine (42). Similar chromatographic systemwas used for quantification of amlodipine andaliskren in tablets (43). Amlodipine, hydrochlorothi-azide and valsartan were analyzed on C18 column inmobile phase system containing acetonitrile, phos-phate buffer at pH 3.7 and triethylamine (44).

Separation of atorvastatin and antihyperten-sive, antidiabetic, antithrombotic drugs in differentcombinations was performed on phenyl column withmobile phase containing addition of 0.1% triethyl-amine acetate to aqueous mobile phase (2). Additionof triethylamine to mobile phase was applied fordetermination of hydrochlorothiazide and inda-pamide in pharmaceutical formulations on C18 col-umn (45).

A lot of analytical methods are applied fordetermination of atorvastatin or amlodipine sepa-rately, but they were rarely determined simultane-ously. Zhou et al. described a method for analysis ofatorvastatin and amlodipine in human plasma sam-ples on CAPCELLPAK CR column with mixed sta-tionary phases: SCX and C18 by use of mobilephase containing acetonitrile and acetate buffer (46).HPLC separation of rosuvastatin, telmisartan, eze-timibe, amlodipine and atorvastatin in pharmaceuti-cal preparations was carried out on C18 column withmobile phase containing mixture of methanol, ace-tonitrile and phosphate buffer at pH 3.0 (24).Amlodipine and atorvastatin in pharmaceutical for-mulations were determined on C18 column withmobile phase containing mixture of methanol, ace-tonitrile and phosphate buffer at pH 5.33 (47).

The aim of this paper was to investigate theretention, peak shape, efficiency, and separationselectivity of selected drugs used to treat hyperten-sion on C18, Phenyl-Hexyl and Polar RP columns.The possibility of the application of DEA as eluentadditive was verified in analysis of acids as well asbases, when amine plays double role as ion pairreagent for acids drugs and silanol blocker (ion sup-presant) for basic drugs. The advantage of the use ofsuch eluent system was tested in analysis of phar-maceutical formulation containing atorvastatin(acid) and amlodipine (base) in one run.

EXPERIMENTAL

Chemicals

The standards of atorvastatin and amlodipinewere obtained from Polfarmex S.A. (Poland) andSandoz (Poland), respectively. Methanol (MeOH)of chromatographic quality, diethylamine (DEA),acetic acid (99-100%), sodium acetate were pur-


Table 3. Values of tR, AS, and N/m for investigated acidic drugs obtained on different columns in eluent system containing MeCN,acetate buffer at pH 3.5 and 0.025 M/L DEA.

C18 Phenyl-Hexyl Polar RP

Name of30% MeCN, acetate 30% MeOH, acetate 30% MeOH, acetate

drugbuffer at pH 3.5 and buffer at pH 3.5 and buffer at pH 3.5 and

0.025 M/L DEA 0.025 M/L DEA 0.025 M/L DEA


Atorvastatin 45.6 2.07 43152 84.53 1.26 45535 75.75 1.28 61212

Fluvastatin 30.69 1.4 61481 66.41 1.18 62780 45.79 1.14 65276

Rosuvastatin 7.01 0.86 19134 13.79 1.14 37520 15.62 1.11 48180

Pravastatin 2.43 0.93 22640 4.03 1.08 25713 4.20 1.05 31189

Valsartan 4.53 1.35 10743 8.16 0.74 10335 9.04 2.10 4777

Telmisartan 27.09 0.7 43616 36.52 0.93 48868 63.23 1.29 63577

Benazapril 5.27 1.64 25575 9.33 1.27 37678 9.45 1.29 42032

Cilazapril 2.96 1.09 38403 5.41 1.21 37940 6.01 1.21 41830

Table 4. Values of tR, AS, and N/m for investigated basic drugs obtained on different columns in eluent system containing MeOH, acetatebuffer at pH 3.5 and 0.025 M/L DEA.

C1850% MeOH, acetate Phenyl-Hexyl Polar RPbuffer at pH 3.5 and 50% MeOH, 50% MeOH,

Name 0.025 M/L DEA acetate buffer acetate buffer of drug * 10% MeOH, acetate at pH 3.5 and at pH 3.5 and

buffer at pH 3.5 and 0.025 M/L DEA 0.025 M/L DEA0.025 M/L DEA


Atenolol * 4.13 0.66 9411 2.09 0.75 9477

Bisoprolol 2.55 0.89 5051

Sotalol * 3.31 0.66 5822

Celiprolol 1.97 0.8 3856 3.48 0.61 6955 5.00 0.78 9561

Ephedrine * 4.29 1.36 1465 2.33 2.41 9601 2.55 0.74 13483

Carvedilol 5.29 1.05 7113 15.18 0.97 18795 42.37 1.24 18259

Caffeine * 6.06 1.67 746 2.93 0.75 9943 7.30 1.14 33278

Losartan 8.25 0.6 1757 18.12 1.01 25610

Nebivolol 13.49 2.46 14503 40.16 0.82 23450 42.10 1.12 25495

Nicergoline 1.88 0.72 4202 4.15 0.60 7855 7.98 1.06 18622

Propranolol 3.42 1.05 6815 7.30 0.85 16310 9.35 0.93 15096

Amlodipine 10.28 2.04 13343 23.88 0.80 20123 27.21 1.13 19489

Diltiazem 5.4 1.19 9406 16.12 0.84 2121 33.73 1.30 25135

Doxazosin 5.97 1.04 9619 14.83 0.64 1889 37.28 1.21 21963

Indapamide 3.45 0.74 4923 7.38 1.18 31496 11.99 1.32 43843

Irbesartan 15.73 5.56 1658 31.84 0.60 15606 50.83 0.96 35857

Clonidine * 6.70 0.7 9630 2.38 0.72 6881 3.11 0.88 10581

Trimetazidine * 28.78 0.94 11425 2.75 0.67 9460 3.77 1.12 19980

Torasemide 3.41 0.73 5388 7.14 0.64 17012 10.65 0.70 23582

Hydrochloro-thiazide

* 5.57 0.64 1389 2.70 0.58 11382

Nimodipine 35.83 1.11 40163 67.43 0.80 26503 153.54 1.88 10128


chased from Merck (Darmstadt, Germany).Pharmaceutical formulation Amlator (10 + 10 mg)was obtained from Gedeon Richter Plc (Budapest,Hungary) Water was double distilled. The acetatebuffer at pH 3.5 was prepared by mixing 0.2 M/Lacetic acid and 0.2 M/L sodium acetate in volumeratio 19 : 1. Drug standards are listed in Table 2.

Apparatus and HPLC conditions

Chromatographic analysis was performedusing liquid chromatograph LaChrom Elite (Merck)equipped with an autosampler, column oven L-7350, solvent degasser L-7612 and DAD detector.The chromatographic measurements were carriedout at 22OC with an eluent flow rate of 1.0 mL/min.The chromatographic separation was performed onKinetex EVO C18 150 mm ◊ 4.6 mm, 5 µm particle

(Phenomenex), Polar RP 80A 150 mm ◊ 4.6 mm, 4µm particle (Phenomenex) and XSELECT Phenyl-Hexyl (150 mm ◊ 4.6 mm, 4 µm) columns. Samples(20 µL) were injected onto HPLC column and elut-ed with mobile phase consisted with methanol (40-55% v/v), acetate buffer ( pH 3.5) 20% v/v, doubledistilled water and 0.025 M/L diethylamine accord-ing to linear gradient elution program (Table 1) TheDAD detector was set in the 200ñ400 nm range andquantitative analysis was performed at 240 nm. Thechromatographic data were acquired and processedwith EZchrom Elite software.

Preparation of stock solutions and calibration

standards

The stock solution of amlodipine and atorvas-tatin at a concentration of 0.2 mg/mL was prepared

Table 5. Values of tR, AS, and N/m for investigated basic drugs obtained on different columns in eluent system containing MeCN, etatebuffer at pH 3.5 and 0.025 M/L DEA.

C1820% MeOH, acetate Phenyl-Hexyl Polar RPbuffer at pH 3.5 and 50% MeOH, 50% MeOH,

Name 0.025 M/L DEA acetate buffer acetate buffer of drug * 5% MeCN, acetate at pH 3.5 and at pH 3.5 and

buffer at pH 3.5 and 0.025 M/L DEA 0.025 M/L DEA0.025 M/L DEA


Atenolol * 4.09 0.69 8515 2.28 4.16 23750 2.49 0.81 23492

Bisoprolol 2.793 1.77 14318

Sotalol * 3.98 0.71 10362 2.28 0.63 17238 2.48 0.81 18158

Celiprolol 2.21 1.44 11463 2.93 0.81 10009 3.59 1.17 16575

Ephedrine * 3.9 2.7 1391 2.26 0.63 15454

Carvedilol 4.44 2.71 7430 16.42 1.74 25964 30.81 2.59 24073

Caffeine *5.62 1.03 5563 2.29 0.73 7422 2.85 1.10 27835

Losartan 12.57 0.64 7971 8.18 0.70 12914 9.68 0.74 37739

Nebivolol 23.77 2.36 21.71 1.44 32016 29.07 1.98 38519

Nicergoline 2.14 1.28 15435 3.18 0.91 14062

Propranolol 4.13 2.3 8682 5.47 1.23 19925 7.13 1.63 21251

Amlodipine 15.92 2.4 9756 13.41 1.35 28336 15.10 1.99 31045

Diltiazem 8.25 2.67 10523 9.58 1.59 29067 15.27 1.97 29799

Doxazosin 7.7 2.53 12894 8.75 1.13 26174 17.30 1.85 37913

Indapamide 13.3 0.82 26836 12.98 0.84 38927 17.61 1.06 65766

Irbesartan 26.42 0.73 24079 12.92 1.10 48338 15.43 0.76 42677

Clonidine * 5.9 0.82 10515 2.28 0.85 2148

Trimetazidine * 22.71 1.44 12170 2.28 0.84 20272 2.48 1.09 17463

Torasemide 3.86 1 23672 4.47 0.75 22810 4.93 0.88 42352

Hydrochloro-thiazide

* 9.94 0.63 9143 3.03 0.62 16062 3.86 0.76 25907

Nimodipine 90.70 0.84 33936 119.58 0.96 4667


by dissolving 10 mg of appropriate drug in 50 mL ofmethanol. The prepared stock solutions were storedat 4OC protected from light. The standard workingsolutions were prepared from the stock solutionsimmediately before the analysis.

Preparation of sample solution

Twenty tablets were weighted and crushed intohomogenous powder. A quantity of powder equiva-lent to one tablet containing 10 mg of amlodipineand 10 mg of atorvastatin was transferred into a 100mL volumetric flask. To this flask, 100 mL ofmethanol were added, and the solution was shakenfor 15 min. From this solution, aliquots of appropri-ate volume were transferred to 10 mL volumetricflasks and diluted to appropriate volume withmethanol.

The standard and sample solutions were deter-mined to be stable for at last 24 h

Method validation

The proposed method was validated withrespect to, linearity, LOD and LOQ, specificity,

accuracy, precision, and robustness. System suit-ability was also evaluated.

System suitabilitySystem suitability was established by injecting

standard solution and results are shown in Tables 2-6.

LinearityAccording to ICH recommendations, at least

five concentrations have to be used for linearity ana-lyzing. In this study seven concentration were cho-sen, in the range from 5 to 100 µg/mL for both drugs(Table 7)

Limit of detection (LOD) and limit of quantifica-tion (LOQ)

LOD and LOQ were calculated according tothe formulas: LOD = 3.3 (SD/S), LOQ = 10 (SD/S),respectively; where SD is the standard deviation ofthe response and S is the slope of the calibrationcurve (Table 7). The calibration curves wereobtained by means of the least square method.

Table 6. Chromatographic parameters obtained on Phenyl-Hexyl column, gradient elution program see Table 1.

Name of drugs tR AS N/m Capacity factor USP resolution

Amlodipine 3.88 1.21 36283 1.38 -

Atorvastatin 8.17 1.13 96871 4.02 17.86

Table 7. Parameters of calibration curves for quantitative analysis of amlodipine and atorvastatin: calibration curves' equations, concen-tration range, regression coefficient (r), limit of detection (LOD), limit of quantitation (LOQ).

Name of Concentrations Equation of calibrationdrug range (µg/mL) curve

r LOD LOQ

Amlodipine 5 - 100 y = 199646x + 265624 0.9996 3.2102 9.7280

Atorvastatin 5 - 100 y = 196225x + 270383 0.9996 3.2750 9.9243

Table 8. Validation data.

Name % of Concentration Intra-day Inter- day

of target added Recovery %RSD Recovery %RSDdrug level (µg/mL) (%) (%)

80 16 101.43 0.48 101.69 0.77

Amlodipine 100 20 100.96 1.48 101.94 1.78

120 24 99.97 0.75 99.69 0.65

80 16 101.47 0.6 101.81 0.85

Atorvastatin 100 20 101.74 1.75 101.98 1.75

120 24 99.81 0.84 99.88 0.65


Accuracy and precision Accuracy of the method was tested by per-

forming recovery studies using the standard additionmethod corresponding to three concentration levels80%, 100% and 120% of each drug. Recoveriesobtained for both drugs are in acceptable range(Table 8).

The precision of the method was assessed bycalculating intra-day and inter-day variation and wasperformed by analyzing six replicates at three con-centrations. In the intra-day studies, drug solutionswere analyzed on the same day and percentage RSDwas calculated. In the inter-day studies, drug solu-tions were analyzed on three consecutive days andpercentage RSD were also calculated.

SpecificityThe chromatograms were checked for the pres-

ence of any extra peaks. Any chromatographic inter-ference from the tablet excipients was detected.Peak purity was also confirmed by comparison ofanalyzed drug UV spectra with the spectra of stan-dards.

RobustnessRobustness was performed by small deliberate-

ly changing chromatographic conditions. Robust-ness was tested by studying the effect of changingtemperature ± 2OC, flow rate ± 0.05 mL/min andwavelengths ± 2 nm. They had not significant effecton the chromatographic parameters like resolutionof the method, peak symmetry, theoretical platesnumber. Small changing of temperature and flowrate also not affect significantly on the peak area.

Application for pharmaceutical preparation

The proposed methods were successfully usedto determine amlodipine, and atorvastatin in theirdosage form ñ Amlator. Five replicate determina-tions were performed. Figure 1 illustrates two chro-matograms obtained following the assay of Amlatortablets (A) and from a standard solution (B). Theresult of the assays undertaken yielded 103.89%(%RSD = 1.32%) and 101.48% (%RSD = 1.49%) oflabel claim for amlodipine and atorvastatin, respec-tively. The observed concentrations of these drugswere found to be 20.78 ± 0.26 µg/mL (mean ± SD)and 20.15 ± 0.3 µg/mL, respectively.


Acidic drugs (Table 2) were chromatographedon C18, Phenyl-Hexyl and Polar RP columns withaqueous mobile phases containing addition of DEA

as ion-pair reagent. Retention times (tR), asymmetryfactors (AS) and theoretical plate numbers per meter(N/m) for investigated drugs obtained on differentcolumns are presented in Tables 2 and 3. The inves-tigated drugs were more strongly retained onPhenyl-Hexyl and Polar RP columns compared toC18 column. This indicated strong interactionsbetween these compounds and π-π stationary phaseligands. On C18 column in eluent system containingmethanol as organic modifier only for two drugssymetrical peaks were obtained, in system withMeCN for five drugs AS values were in optimalrange. More symmetrical peaks were obtained oncolumns with π-π ligands - Phenyl-Hexyl and PolarRP. On Phenyl-Hexyl column for 6 out of 8 drugs ineluent containing MeOH and for 7 out of 8 in eluentwith MeCN symmetrical peaks were obtained. OnPolar RP column only for pravastatin in MeOH andfor valsartan in MeCN as organic modifiers asym-metrical peaks were observed. Higher values of N/mwere obtained on all tested columns for all investi-gated compounds in systems containing MeCN asorganic modifier. Great differences in system effi-ciency were obtained on different columns e.g., foratrovastatin N/m obtained on C18 column in systemwith MeOH was 7290, on Phenyl-Hexyl 17770 andon Polar RP 18980; for rosuvastatin N/m obtainedon C18 column in system with MeCN was 19130,on Phenyl-Hexyl 37520 and on Polar RP 48180. Thehighest N/m values for most investigated drugs wereobtained on columns with π-π ligands, especially onPolar RP column.

In the next series of experiments basic drugstandards (Table 4) were chromatographed on alkyl-bonded, Phenyl-Hexyl and Polar RP columns in elu-ent systems containing methanol (MeOH) or ace-tonitrile (MeCN) as organic modifiers, acetatebuffer at pH 3.5 and diethylamine (DEA). Thesechromatographic systems were compared in termsof retention of acidic or basic drugs on different sta-tionary phases that were selected according to theirpotential differences in retention mechanism andhence possible changes in performance, peak shapeand selectivity. Retention, selectivity and systemsefficiency obtained on three tested columns in eluentsystems with MeOH or MeCN as organic modifiersobtained for basic drugs were compared (Tables 4and 5). Different retention, selectivity and systemsefficiency were obtained on different columns withmobile phases containing MeOH or MeCN asorganic modifiers. The better system efficiency andmore symmetrical peaks for most investigated drugswere obtained on Phenyl-Hexyl or Polar RPcolumns compared to those obtained on C18 column


in booth eluent systems. In systems with eluentscontaining MeOH most symmetrical peaks were onPolar RP column, but when MeCN was added tomobile phases optimal AS values (0.8 < AS < 1.5)were obtained on Phenyl-Hexyl column for mostinvestigated drugs. The highest N/m values for mostcompounds were obtained in both eluent systems onPolar RP column. In chromatographic system con-taining MeOH for 7 and in system with MeCN for13 drugs N/m > 20 000.

Most selective and efficient system was chosenfor separation of amlodipine and atorvastatin inpharmaceutical formulation Amlator. For most

investigated drugs more symmetrical peaks andhigher N/m values were obtained for systems withaddition of MeCN. For this reason, eluent contain-ing MeCN was chosen for determination of Amlatorcomponents. Taking into account peak symmetry,better on Phenyl-Hexyl column eluted with mobilephase containing MeCN, determination of Amlatorcomponents was performed on Phenyl-Hexyl col-umn with MeCN as organic modifier. For reductionof analysis time gradient elution mode was appliedfor simultaneous separation of these drugs in thepharmaceutical formulation. Chromatographicparameters obtained in the system are presented in

Figure 1A. Chromatogram obtained for separation of atorvastatin (20 µg/mL) and amlodipine (20 µg/mL) standard solution on Phenyl-Hexyl column. Gradient elution program see Table 1


Table 6. Drugs were separated in times less than 10min, obtained peaks were symmetrical and systemefficiency was high.

Typical chromatogram obtained for atorvas-tatin and amlodipine standards solution at 20 µg/mLconcentration is presented in Figure 1A and for sep-aration of Amlator components is presented inFigure 1B.

The quantitative analysis was performed by acalibration curve method. Table 7 presents parame-ters of the calibration curves for the investigateddrugs. The identities of analyte peaks in pharmaceu-tical formulation samples were confirmed by com-

parison of their UV spectra with the spectra of drugstandards. LODs obtained by the method were3.2102 µg/mL and 3.2750 µg/mL and LOQs were9.7280 µg/mL and 9.9243 µg/mL for amlodipine andatorvastatin, respectively. Proposed method wasvalidated, parameters are presented in Table 8. Intra-day recovery were obtained in range 99.81-101.74%and inter-day recovery were from 99.88 to 101.98%.

CONCLUSIONS

The changes of stationary phases and organicmodifiers in mobile phase enables on variations in

Figure 1B. Chromatogram obtained for separation of atorvastatin and amlodipine in Amlator on Phenyl-Hexyl column. Gradient elutionprogram see Table 1


retention of acidic or basic compounds, separationselectivity, peaksí symmetry and systems efficien-cy. More symmetrical peaks and higher N/m valuesfor most investigated drugs were obtained in chro-matographic systems with acetonitrile as modifier ofmobile phases.

The separation selectivity, peaks symmetryand system efficiency on C18 column were worsecompared to Phenyl-Hexyl and Polar RP columns inboth eluent systems for most acidic and basic drugs.

The obtained results indicate that the stationaryphases containing ligands with π-π electrons com-pared to C18 stationary phase offer better peaksshape and system efficiency for most investigatedionizable drugs.

The simultaneous determination of atorvastatinand amlodipine in pharmaceutical formulation byHPLC was performed on Phenyl-Hexyl column withmobile phase containing mixture of acetonitrile,acetate buffer at pH 3.5 and 0.025 M/L DEA.Proposed method was validated.

The application of stationary phases with π-πmoieties and the addition of DEA to mobile phaseslet to obtain good peaksí symmetry and high systemefficiency because of double protection againstinteraction between basic compounds and freesilanol groups or creation of ion-pair between acidiccompounds and DEA.

Optimization of retention behavior of the inves-tigated drugs enables a choice of the best chromato-graphic system containing DEA which was applied asion-pair or silanol blocker reagent for simultaneouslydetermination of acidic drug atorvastatin and basicdrug amlodipine in pharmaceutical formulation.

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Received: 3. 12. 2015


Eplerenone (EPL), a selective aldosteronereceptor antagonist, is indicated in the treatment ofchronic heart failure and hypertension. The drugwas launched in 2002 and in order to allow its wideruse new generic drugs needed to be developed astheir clinical equivalence to the brand-name cardio-vascular drugs was confirmed in the recent meta-analyses (1). One of the important steps in genericdrug development is a bioequivalence study basedon pharmacokinetic endpoints which requires a val-idated bioanalytical method to evaluate drug con-centration in a human body fluid, usually plasma.

There are only a few published methods for thedetermination of eplerenone in human plasma orserum (2-5). Among them, a high-performance liq-uid chromatography (HPLC) coupled to mass spec-trometry (2-4) is the most commonly applied tech-nique. The only reported method using HPLC withultraviolet detection (5) is not sensitive enough forbioequivalence studies. Maximum plasma concen-tration values reported (6) suggest the possibility ofeplerenone determination in human plasma after a

single oral administration of 25 or 50 mg dose usinga single quadrupole MS detection (4).

The reliability of a bioanalytical method and itscompliance with regulatory guidelines cannot becompromised because measured drug concentra-tions are the basis for the conclusions regarding drugefficacy and patient safety. However, some conceptsof green analytical chemistry may be applied with-out any loss of the resultsí reliability, including thesubstitution of toxic reagents, minimalization ofsample volume and reduction of waste volume (7-9). It should be noted that during a bioequivalencestudy a considerable number of samples ñ usuallyover 1000, is analyzed, which makes every attemptto reduce harm caused to the environment by ana-lytical processes even more important. Among thepreviously reported LC/MS methods it is hard tofind a green assay. Zhang et al. (2) applied solidphase extraction (SPE) which generates unnecessarywaste of cartridges and used acetonitrile in themobile phase which could be replaced by methanol.In the other two methods the sample preparation by

ENVIRONMENTALLY FRIENDLY LC/MS DETERMINATION OF EPLERENONE IN HUMAN PLASMA

KATARZYNA BUå-KWAåNIK*,# MONIKA FILIST#, PIOTR J. RUDZKI

Pharmaceutical Research Institute, Pharmacology Department, 8 Rydygiera St., 01ñ793 Warszawa, Poland

Abstract: Eplerenone (EPL), a selective aldosterone receptor antagonist, is indicated in the treatment of chron-ic heart failure and hypertension. It is hard to find a green assay among a few published methods for its deter-mination in human plasma or serum. Following a liquid-liquid extraction with methyl t-butyl ether, eplerenoneand isotope labelled eplerenone ñ used as an internal standard ñ were separated from the endogenous com-pounds on an Atlantis dC18 column (150 ◊ 3 mm, 3.0 µm). An isocratic elution with the mobile phase con-sisting of methanol and ammonium acetate (3 : 2, v/v) was used. A single quadrupole mass spectrometer wasoperated in positive electrospray ionization using the selected ion monitoring mode. The method is more envi-ronmentally-friendly than the previously reported assays. Acetonitrile in the mobile phase was replaced withmethanol which is a removable solvent. Plasma sample volume was reduced to 250 µL which significantlydecreased waste volume. Chlorinated solvents used previously for liquid-liquid extraction were eliminated andthe safety of the laboratory staff was increased by eliminating diethyl ether. The method is characterized by ashort analysis time, simple sample preparation and reduction of waste volume, which are important advantageswhen analyzing large numbers of samples. The method was validated according to international regulatoryguidelines and may be applied to human pharmacokinetic studies following a single 25 or 50 mg oral dose.

Keywords: eplerenone, high-performance liquid chromatography-mass spectrometry, bioanalytical methodvalidation, liquid-liquid extraction, pharmacokinetics, green analytical chemistry

1487

* Corresponding author: e-mail: [email protected]. # Katarzyna Buú-Kwaúnik and Monika Filist contributed equally to this work

1488 KATARZYNA BUå-KWAåNIK et al.

liquid-liquid extraction could be more environmen-tally-friendly: Almeida et al. (3) used a chlorinatedsolvent, while Quian et al. applied 3 mL of an organ-ic solvent per sample (4).

The aim of this study was to to develop andvalidate an environmentally-friendly method foreplerenone determination in human plasma by (A)using greener HPLC solvents, (B) reducing samplevolume and the amount of organic solvents usedduring liquid-liquid extraction as well as (C) avoid-ing unnecessary waste such as SPE cartridges.

EXPERIMENTAL

Chemicals and reagents

Reference standards: eplerenone, (7α,11α,17α)-9,11-epoxy-17-hydroxy-3-oxopregn-4-ene-7,21-dicarboxylic acid as γ-lactone methyl ester (CASnumber: 107724-20-9) was synthesized at thePharmaceutical Research Institute (Warsaw,Poland), eplerenone-d6 (the internal standard, IS)and eplerenone hydroxyacid (eplerenone metabolite)were supplied by TLC Pharmachem (Vaughan,Ontario, Canada), 6β-hydroxyeplerenone (epleren-one metabolite) was supplied by Molcan Corporation(Toronto, Ontario, Canada). Methanol and acetoni-trile (both LC-MS grade), methyl t-butyl ether(HPLC grade) were obtained from POCH (Gliwice,Poland). The ammonium acetate (analytical grade)was purchased from Avantor (Gliwice, Poland).Purified water from Milli-Q system, Millipore(Molsheim, France), was used throughout the study.

Instrumentation

The Shimadzu LC/MS system (Duisburg,Germany), consisting of two LCñ10ADVP pumps,an SILñHTA autosampler, a CTOñ10A columnoven, a DGUñ20A3 degasser and an LCMSñ2010single quadrupole mass spectrometer, was used foreplerenone determination in human plasma. Dataintegration was performed with the ShimadzuísLCMS Solution software (version 2.05).

Chromatographic conditions

The chromatographic separation from theendogenous compounds was performed on anAtlantis dC18 column (150 ◊ 3 mm, 3.0 µm) fromWaters (Milford, MA, USA). The HPLC column waspreceded by a SecurityGuard C-18 guard column (4 ◊3 mm) purchased from Phenomenex (Torrance, CA,USA). The mobile phase, consisting of methanol and10 mM ammonium acetate in the ratio 3 : 2, v/v, wasdelivered at the flow rate of 0.3 mL/min. The samplevolume of 5 µL was injected onto the column. The

column was maintained at 45 ± 2OC. The total chro-matographic run time was 7.0 min with the retentiontime of eplerenone approximately 5.2 min.

Mass spectrometric conditions

The mass spectrometer was equipped with anelectrospray ionization source operated in the positivemode, using single ion monitoring (SIM) as the dataacquisition mode. The ions of eplerenone and the ISwere monitored at the m/z ratio of 415.0 and 418.0,respectively. The ions of eplerenone metabolites:eplerenone hydroxyacid and 6β-hydroxyeplerenonewere monitored at the m/z ratio of 433.0 and 431.0,respectively. The probe high voltage was 4.5 kV andnitrogen at 4.0 L/min was used as the nebulizer gas.The block temperature was 250OC, whereas the curveddesolvation line temperature and voltage were 280OCand 0 V, respectively. The detector voltage was 1.5 kV.

Stock and working solutions

Stock solutions of eplerenone, eplerenonemetabolites and the IS were prepared by dissolvingappropriate amounts of the substances in methanol.The stock solutions were further diluted with 50%acetonitrile (v/v) to the working solutions. Theworking solutions used to prepare the calibrationstandards and quality control samples were obtainedfrom different stock solutions. The stock solutionsof eplerenone and the IS were stored in a freezer atñ10OC temperature and the working solutions werestored in a refrigerator at 10OC temperature. Thestock and working solutions of eplerenone metabo-lites were prepared ex tempore.

Calibration standards and quality control sam-

ples

The calibration standards contained eplerenoneat the following concentration levels: 25, 50, 250,500, 1000, 1250, 1500 and 2000 ng/mL. The quali-ty control samples contained eplerenone at the con-centration levels: 50, 1000 and 1500 ng/mL. Thecalibration standards and quality control sampleswere prepared by spiking blank human plasma withthe appropriate working solution of eplerenone. Thecalibration standards were prepared directly beforethe analysis and the quality control samples werestored in a freezer at ñ20OC temperature.

Sample preparation

Twenty five µL of the IS solution was added tothe 250 mL aliquot of human plasma and vortexmixed in a glass, screw cap extraction tube. Then 1mL of methyl t-butyl ether was added and shaken ona vibrax mixer for 10 min. After centrifugation, the

Environmentally friendly LC/MS determination of eplerone in human plasma 1489

aqueous phase was frozen and the organic layer wastransferred to a glass tube and evaporated to drynessunder the stream of nitrogen. The dry residue wasreconstituted in 200 mL of 50% acetonitrile (v/v),mixed and transferred into an autosampler vial.

Method validation

The validation parameters were defined accord-ing to the European Medicines Agency as well as theFood and Drug Administration guidelines (10-12).Validation tests included linearity, the lower limit ofquantification (LLOQ), selectivity, matrix effect,recovery, the within-run (within one analyticalsequence) and between-run (within three analyticalsequences) accuracy and precision, dilution integrity,carry over, as well as the stability evaluation.

For the calculation of the precision, accuracy,calibration curve parameters and selected stabilityresults, a normal distribution of measurements wasassumed. The confidence intervals (CIs) were appliedfor the statistical analysis of the stability (13). Possibleinterferences from the endogenous compounds wereevaluated by analyzing blank human plasma samplesderived from six sources, including hemolyzed andhyperlipidemic plasma. Moreover, the influence ofmajor plasma metabolites: eplerenone hydroxyacidand 6β-hydroxyeplerenone, was studied in the con-centration of 60 and 260 ng/mL, respectively (14).Testing for metabolite influence on determination ofthe parent drug is important to increase the chance tomeet criteria of ìincurred sample reanalysisî duringthe pharmacokinetic study (15). The interferences ofthe commonly used analgesic drugs ibuprofen andparacetamol in the concentrations of 50 and 20 µg/mL,respectively, were tested as described by Filist et al.(16). The matrix factors (11) were calculated at threelevels (50, 1000, 1500 ng/mL) and for the IS at work-ing concentration. The IS-normalized matrix factorand relative matrix effect in different sources, includ-ing hemolyzed and hyperlipidemic plasma, should notexceed 15%.

RESULTS

Method development

Eplerenone is chemically and enzymaticallyconverted to an open lactone form, but no intercon-version is observed at pH between 6.5 and 8 (2).Therefore, a neutral pH was applied in the mobilephase and during sample preparation. To assure thestability of eplerenone in the plasma samples nomodification of pH was necessary contrary to ourexperience with temozolomide which required acid-ification of the human plasma to pH below 4 (17).

To avoid increased energy consumption by LC-MS, the initial experiments were performed usingHPLC with a UV detection and the mobile phase asdescribed by Gide et al. (5). Chromatographic condi-tions were optimized, by injecting eplerenone stan-dard onto a Zorbax SB-C18 analytical column (150◊ 3.0 mm; 3.5 µm). During the isocratic elution with50% acetonitrile (v/v), eplerenone signal wasobserved very close to the baseline disturbance asso-ciated with the column dead time (2.2 min), while thesignal of exemestane, used as the IS, was at 13.5 min.Therefore, the organic component was reduced to40% and the IS was changed to anastrozole. The dis-tance between the two signals was unsatisfactoryeven after further modifications to the mobile phasecomposition, so gradient elution was applied and theIS was changed back to exemestane. After theextraction of the plasma samples, peaks derived fromthe endogenous compounds were observed in theretention times of both the analyte and the IS.Despite the changes in the gradient program, thecomposition of the mobile phase, wavelength and thetype of the chromatographic column, required selec-tivity was not achieved.

In order to eliminate the interferences of theendogenous compounds, a UV detector wasreplaced with the mass spectrometer, but to avoidunnecessary energy consumption a single instru-ment instead of a tandem quadrupole was tested. Anisotope labelled IS (eplerenone-d3) was used tomeet regulatory recommendations (11). To makeour method more environmentally-friendly, acetoni-trile was substituted with methanol in the mobilephase, which in turn increased the signal intensityfor eplerenone. Among the columns tested, the bestsensitivity with a suitable retention time wasobtained on an Atlantis dC18 column (150 ◊ 3 mm,3 µm).

The optimization of detection conditionsincluded the selection of positive ion electrosprayfor all detected compounds. The mass scan spectraof eplerenone and the IS showed that the molecularions [M + H]+ of m/z 415.0 and m/z 418.0, respec-tively, were the most abundant. Eplerenone and theIS were monitored in all measurements throughoutthe study, while the metabolites, i.e., eplerenonehydroxyacid (m/z 433.0) and 6β-hydroxyeplerenone(m/z 431.0), were monitored only during the selec-tivity test.

The sample preparation was optimized to providea good clean-up and repeatable recovery using a sim-ple procedure. During the experiments using the UVdetection, a 500 µL of plasma per sample was neces-sary, while after turning to the MS detection this vol-


ume was reduced by half. Both protein precipitation(by methanol and acetonitrile) and liquid-liquid extrac-tion were tested. The extracting solvents tested at 3 mLper sample included: methyl t-butyl ether (MTBE),ethyl acetate and dichloromethane : diethyl ether mix-ture (4 : 6, v/v). MTBE led to the best recovery andsample clean-up, while other procedures yielded lowextraction efficiency and the appearance of peaks fromthe matrix in the eplerenone retention time. Finally, thevolume of MTBE was reduced to 1 mL.

Initially, 50% acetonitrile (v/v) was used for thereconstitution of the dry residue obtained followingevaporation. During the method development themixtures of acetonitrile and water in the concentra-tions of 20, 30, 40 and 80% (v/v) were tested. Thesamples reconstituted with 20% acetonitrile con-tained an insoluble residue, 30% and 40% (v/v) wereinadequately cleaned, whereas 80% acetonitrile (v/v)caused the deterioration of a peak shape. Finally, opti-mized sample preparation conditions led to the highrecovery of both eplerenone and the IS (73-79%).

Method validation

All experiments were conducted in compliancewith the principles of Good Laboratory Practice(GLP, certificate 1/2014/DPL issued by the Bureaufor Chemical Substances, £Ûdü, Poland).

LinearityThe calibration curve was linear within the

range of 25-2000 ng/mL. A weighted linear regres-

sion analysis with the weighting factor of 1/y2 select-ed according to the minimum sum of percentage rel-ative errors was applied (18). The values of theregression parameters for the curve, described by theequation: y = ax + b, were: a = 0.00170, b = 0.0042and r = 0.9994 (n = 6), where x was the eplerenonenominal concentration, y was the eplerenone/IS peakarea ratio, a and b were slope and intercept, respec-tively. All regression parameters were statisticallysignificant (significance level 0.05, df = n ñ 2).

SelectivityTo confirm the method selectivity, blank human

plasma from six different sources, including hemolyzedand hyperlipidemic plasma, was analyzed. The chro-matograms showed no peaks influencing the quantifi-cation near the retention times of eplerenone and the IS(Fig. 1). The accuracy of the method at the QC samplesspiked with eplerenone hydroxyacid and 6β-hydroxye-plerenone was in the range of 85-115%, therefore theinfluence of eplerenone major metabolites on the deter-mination of eplerenone was insignificant. For the QCsamples containing ibuprofen and paracetamol theaccuracy was within the acceptance criteria, which con-firmed the lack of the influence of ibuprofen and para-cetamol on the quantification of eplerenone.

Dilution integrity and carry-overDilution integrity was confirmed at 3000 ng/mL

after the dilution with blank human plasma (1:1, v/v).The accuracy and precision were 107.5-109.1% and

Table 1. Validation summary.

Linear range [ng/mL] 25 - 2000

Eplerenone concentration [ng/mL] 25 50 250 1500

Selectivity with eplerenone metabolites precision [%]*

- 0.52-1.24 - 0.76-1.82

Selectivity with eplerenone metabolites accuracy [%]*

- 106.3-107.5 - 107.3-109.1

Selectivity with ibuprofen and paracetamol precision [%]*

- 1.41-3.38 - 0.86-2.06

Selectivity with ibuprofen and paracetamol accuracy [%]*

- 102.4-105.7 - 108.1-110.2

Extraction recovery of eplerenone [%]

- 72.7 74.1 79.3

Matrix effect - RSD [%] - 0.44 0.64 0.45

Within-run precision [%]* 0.84-2.02 1.01-2.41 3.47-8.30 1.56-3.74

Within-run accuracy [%]* 101.9-103.8 101.5-103.8 100.5--108.6 107.9-111.7

Between-run precision [%]* 3.09-4.84 2.19-3.43 2.38--3.73 1.63-2.55

Between-run accuracy [%]* 103.8-107.1 104.7-107.0 103.6-106.1 109.2-110.9

*The accuracy and precision results are expressed as 90% confidence intervals.


0.66-1.59%, respectively (results expressed as 90%CIs for the percentage of the nominal value and CV,respectively). The carry-over experiment, in whichblank human plasma samples were analyzed immedi-ately after the highest concentration calibration stan-dards (procedure repeated six times), showed nopeaks influencing the quantification.

Matrix effect and recoveryThe matrix effect was evaluated from the RSD

of the IS-normalized matrix factor (i.e., the ratio ofthe matrix factors calculated for eplerenone and theIS) calculated for six different sources of plasma,including hemolyzed and hyperlipidemic plasma.The calculated RSD did not exceed 15%, which con-firmed the absence of matrix effects (Table 1).

The calculation of the eplerenone and IS recov-ery was based on the ratio of the peak areas deter-mined in the pre- and post-extraction spiked plasmasamples from six different sources, includinghemolyzed and hyperlipidemic plasma (19). Theextraction recovery of eplerenone was stable acrossthe studied concentration range and did not influ-ence the recovery of the IS (Table 1).

Accuracy and precisionThe results of the within-run (one sequence, n

= 6 for each concentration) and between-run (three

sequences, n = 6 for each concentration) accuracyand precision at the LLOQ (25 ng/mL) and at threeQC levels met the acceptance criteria (Table 1):90% CIs for the percentage of the nominal valuewere within 80-120% for LLOQ and 85-115% forQCs; the 90% CIs for CV were below 20% forLLOQ and below 15% for QCs.

StabilityFor each tested solution 90% CI of the mean

stability met the acceptance criteria, falling withinthe range of 90-110% (Table 2). The results of thestability tests in the plasma samples included fol-lowing tests: autosampler, freeze and thaw, short-term and long-term at two storage temperatures for14 days. The acceptance criteria were met for eachconcentration level 90% CI for the mean stabilityfell within the 85-115% (Table 3).

DISCUSSION AND CONCLUSIONS

A novel method for eplerenone determination inhuman plasma was developed and validated accord-ing to international regulatory guidelines (11, 12).The method is more environmentally-friendly thanpreviously reported assays. First of all, acetonitrile inthe mobile phase (2) was replaced with methanolwhich is a removable solvent. To avoid unnecessary

Table 2. The stability of eplerenone in plasma expressed as 90% confidence intervals (n = 6).

Stability Short-term Freeze-thaw Autosampler Long-term Long-term

TemperatureAmbient Ambient

temperature-20OC

temperature-20OC -70OC

Storage period 4 h 3 cycles 69 h 14 days 14 days

Eplerenone concentration

50 95.4-98.3 94.6-100.6 101.2-102.6 106.6-110.8 105.5-109.7

[ng/mL] 1500 94.5-97.8 93.3-97.1 96.1-96.8 101.5-106.1 101.7-105.9

Table 3. The stability of eplerenone and the IS in the solutions expressed as 90% confidence intervals (n = 6).

Analyte Concentration Temperature Storage period Stability [%]

-10OC 85 days 100.9-106.2

Stock Eplerenone 1.00 mg/mL

Ambient temperature 24 h 99.2-102.5

solutions -10OC 32 days 96.6-105.6IS 0.10 mg/mL


10OC 9 days 99.8-102.6

Eplerenone

100 µg/mLAmbient temperature 24 h 99.6-104.4

Working 10OC 9 days 98.5-100.7

solutions250 ng/mL


IS 6.5 µg/mL10 C 9 days 96.7-99.8



waste of SPE cartridges, the samples were preparedby liquid-liquid extraction. Plasma sample volumewas reduced to 250 µL ñ the lowest value among themethods reported for eplerenone determination,which allowed to reduce organic solvent consumptionfrom 3 mL (4) or 5 mL (5) to as little as 1 mL persample. The reduction in the plasma sample volumesignificantly decreased the waste volume generatedby the method. Moreover, chlorinated solvents usedpreviously for liquid-liquid extraction, i.e., 1-chlorobutane (3) and dichloromethane (5), were elim-inated and the safety of the laboratory staff wasincreased by eliminating diethyl ether (5).

Although there are some metrics proposed toevaluate how green is a particular analytical method(7-9), they are difficult for application to the bioan-alytical methods. For example the Eco-Scale pre-sented by Ga≥uszka et al. gives penalty points for the

use of reagents in the following volumes: below 10mL, 10-100 mL and over 100 mL. Those volumesare not applicable to the bioanalytical methods usedin the pharmacokinetic studies where use of solventsis usually below 5 mL per sample. However, inthose studies ñ taking into account considerablenumber of samples ñ reduction of each 1 mL persample means reduction of hundreds or thousands ofmL per study. This observation indicates the need todevelop metrics dedicated for green bioanalyticalmethods.

Tandem mass spectrometry detectors are cur-rently regarded as a standard in pharmacokineticstudies, as they assure selectivity and sensitivity inthe complex sample matrices like blood, serum,plasma or urine (20, 21). Single quadrupole detec-tors are a more cost-effective option and they aregreener due to a lower energy consumption. As

Figure 1. Selectivity ñ no interfering peaks were recorded on the MS chromatograms of blank human plasma (N ñ normal, L ñ hyperlipi-demic, H ñ hemolyzed) for eplerenone (top) and the internal standard (IS, bottom). The MS chromatogram of the LLOQ at 25 ng/mLwas added for comparison


illustrated by the present paper and previousresearch (22-24), a single quadrupole mass spec-trometer with an appropriate sample preparation isable to detect the analyte in ng/mL concentrationsand is still an appropriate tool for bioanalysis.

The method is characterized by a short analysistime, simple sample preparation and reduction ofwaste, which are important advantages when ana-lyzing large numbers of samples. The assay wasfully validated with respect to the EuropeanMedicines Agency and the Food and DrugAdministration requirements, proving its reliability,and may be applied to pharmacokinetic studies inhumans following a single 25 or 50 mg oral dose.

Acknowledgments

The present study was supported by theEuropean Union (European Regional DevelopmentFund) under the Innovative Economy OperationalProgramme 2007-2013 (project no. UDA-POIG.01.03.01-14-062/09). The sponsor was notinvolved in conducting the study. The authors aregrateful to Dr. Jacek Musijowski for his scientificcontribution during the method development andMrs. Krystyna Serafin-Byczak for her excellenttechnical assistance. The authors wish to thank Dr.Micha≥ ChodyÒski and his synthetic and analyticalteam from the Chemistry Department and R&DAnalytical Chemistry Department of Pharmace-utical Research Institute (Warsaw, Poland) for sup-plying the eplerenone reference standard.

REFERENCES

1. Manzoli L., Flacco M.E., Boccia S., DíAndreaE., Panic N. et al.: Eur. J. Epidemiol. 31, 351(2015).

2. Zhang J.Y., Fast D.M., Breau A.P.: J.Chromatogr. B 787, 333 (2003).

3. Almeida S., Pedroso P., Filipe A., Pinho C.,Neves R. et al.: Arzneimittelforschung 61, 153(2011).

4. Qian W.J., Ding L., Wen A.D., Gong B., LengY. et al.: Acta Pharm. Sin. 44, 771 (2009).

5. Gide P., Sonawane S., Chitnis A.: J. Pharm.Anal. 2, 390 (2012).

6. Public Assessment Report DK/H/1988/001-002/DC http://www.pdfoz.net/k-20210502.html(accessed on 07. 07. 2016).

7. Ga≥uszka A., Konieczka P., Migaszewski Z.M.,Namieúnik J.: Trends Anal. Chem. 37, 61(2012).

8. Ga≥uszka A., Migaszewski Z., Namieúnik J.:Trends Anal. Chem. 50, 78 (2013).

9. Tobiszewski M., MarÊ M., Ga≥uszka A.,Namieúnik J.: Molecules 20(6), 10928(2015)

10. Guideline on the Investigation of Bioequi-valence. Committee for Medicinal Products forHuman Use (CPMP/EWP/QWP/1401/98/ Rev.1 Corr.**). London, 20 January, 2010.

11. Guideline on bioanalytical method validation.European Medicines Agency (EMEA/CHMP/EWP/192217/2009 Rev.1 Corr. 2**). London,21 July, 2011.

12. Guidance for Industry. Bioanalytical MethodValidation. U.S. Department of Health andHuman Services. Food and DrugAdministration. Center for Drug Evaluation and Research (CDER). Center for VeterinaryMedicine (CVM). May 2001.

13. Rudzki P.J., Leú A.: Acta Pol. Pharm. Drug Res.65, 743 (2008).

14. Clinical Pharmacology and BiopharmaceuticsReview, Inspra 25, 50, 100 mg film-coatedtablets; application number: 21-437/S-002http://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/21437S002_Inspra_BioPharmr.pdf(accessed on 11. 07. 2016).

15. Kaza M., Gilant E., Filist M., Szlaska I.,PawiÒski T., Rudzki P.J.: Clin Biochem. 47,1313 (2014).

16. Filist M., Szlaska I., Kaza M., PawiÒski T.:Biomed. Chromatogr. 30, 953 (2016).

17. Gilant E., Kaza M., Szlagowska A., Serafin-Byczak K., Rudzki P.J.: Acta Pol. Pharm. DrugRes. 69, 1347 (2012).

18. Almeida A.M., Castel-Branco M.M., Falc„oA.C.: J. Chromatogr. B 774, 215 (2002).

19. Matuszewski B.K., Constanzer M.L., Chavez-Eng C.M.: Anal. Chem. 75, 3019 (2003).

20. DereziÒski P., KlupczyÒska A., Sawicki W.,Kokot Z.: Acta Pol. Pharm. Drug Res. 73, 303(2016).

21. Want E., Compton B.J., Hollenbeck T., SiuzdakG.: Spectroscopy 17, 681 (2003).

22. Musijowski J., Filist M., Rudzki P.J.: Acta Pol.Pharm. Drug Res. 71, 1029 (2014).

23. Musijowski J., PiÛrkowska E., Rudzki P.J.: J.Sep. Sci. 37(19), 2652 (2014).

24. KsyciÒska H., Rudzki P.J., Sarosiek A.: ActaPol. Pharm. Drug Res. 63, 417 (2006).

Received: 19. 07. 2016


Imatinib mesylate belongs to the group of theprotein tyrosine kinase inhibitors and platelet-derived growth factor receptors. It is mainly indicat-ed for the treatment of chronic myeloid leukemia andgastrointestinal stromal tumor (1). Imatinib is metab-olized in the liver to at least thirteen metabolites (2).The main pharmacologically active metabolite ñ N-desmethyl imatinib ñ reaches ca. 10% of the parentdrug concentration in plasma (1, 3, 4). The mean val-ues of the maximum plasma concentration followinga 400 mg single oral administration range from 1600to 3000 ng/mL and are reached within ca. 3 h (1, 5-7). N-desmethyl imatinib reaches the maximum plas-ma concentration at a similar time but its mean plas-ma half-life of ca. 40 h is 2.5 times longer than thehalf-life of imatinib (4).

The purpose of bioequivalence studies is tocompare reference and generic medicinal products.Therefore, the studies are designed to distinguish theformulation effect from other effects and generally,the drug is administered in a single dose to thehealthy volunteers who do not take any other drugsduring the study. In order to evaluate bioequiva-lence, only the parent compoundís determination isrequired, as the studyís aim is to compare the for-

mulations and not differences in the metabolismbetween subjects. The metabolites do not need to bequantified (7) but assessing their influence on themethodís reliability is still important. In the report-ed high-performance liquid chromatography meth-ods with ultraviolet detection (HPLC-UV) N-desmethyl imatinib was either quantified (8-13)) orchromatographically separated from the peak of theparent compound (14, 15). However, chromato-graphic separation may not be enough to make surethat the main metabolite does not influence thequantification of the analyte. Therefore, the risk ofN-desmethyl imatinibís back-conversion to the par-ent drug during sample preparation and storage wasevaluated in this study.

Numerous methods for the imatinib quantifica-tion in human plasma are based on liquid chro-matography coupled with mass spectrometric detec-tion. Miura and Takahashi (16) listed 22 of those butthere is a few more (4-6, 17, 18). These methods arerapid and highly sensitive, up to 1 ng/mL (2, 18).However, the lower limit of quantification (LLOQ)of 40 ng/mL should enable us to determine ima-tinibís pharmacokinetic profile in a bioequivalencestudy of two imatinib medicinal products following

HPLC-UV ASSAY OF IMATINIB IN HUMAN PLASMA OPTIMIZED FORBIOEQUIVALENCE STUDIES

MICHA£ KAZA*, EDYTA PI”RKOWSKA, MONIKA FILIST and PIOTR J. RUDZKI

Pharmaceutical Research Institute, Pharmacology Department, 8 Rydygiera St., 01-793 Warszawa, Poland

Abstract: Imatinib is an anticancer drug approved for the treatment of a number of cancers, mostly used inchronic myeloid leukemia. Numerous bioanalytical methods using high performance liquid chromatographycoupled to ultraviolet detection point at the importance and necessity of the therapeutic drug monitoring of ima-tinib. Unfortunately, these methods are not optimized for single dose pharmacokinetic studies such as bioe-quivalence. In this study, attention was turned mostly to the analysis time, linearity range and interferences byendogenous components of the matrix and exogenous substances - especially metabolites. The method enablesthe quantification of imatinib in the presence of the main metabolite (N-desmethyl imatinib). Its potential back-conversion was examined during storage for 4 h at ambient temperature as well as for 239 days at -20∞C. Thesample preparation based on the liquid-liquid extraction was combined with a short analysis time of 7 min.Therefore, the method was suitable for analyzing large numbers of samples in a short time. The linearity rangeof 40-4000 ng/mL was optimized for human pharmacokinetic studies after a single 400 mg oral dose of ima-tinib. Successful application in a bioequivalence study confirmed the reliability of the method.

Keywords: N-desmethyl imatinib, metabolite back-conversion, high performance liquid chromatography, phar-macokinetics, bioanalytical method validation

1495


1496 MICHA£ KAZA et al.

the administration of a 400 mg single oral dose.Desired sensitivity may be obtained using a moreaccessible and cost-efficient UV detection. Miuraand Takahashi (17) listed 12 HPLC-UV methods forthe quantification of imatinib, but none of them hasbeen applied to a bioequivalence study. The bioana-lytical methods used in bioequivalence studies haveto be: (a) optimized regarding to the linarity rangeand (b) rapid and robust to analyze a large numberof samples in a short time. Most HPLC-UV methodsare thus unsuitable for a bioequivalence study due toa long analysis time exceeding 30 min (9-11, 19, 20)or an inappropriate calibration range (11- 20-22).

The aim of the study was to optimize for abioequivalence study and validate a simple andrapid HPLC-UV method for imatinib determinationin human plasma. The influence of the main metabo-lite (N-desmethyl imatinib) on the quantification ofthe parent drug substance was also assessed.

EXPERIMENTAL

Chemicals and reagents

Imatinib mesylate (CAS No. 220127-57-1, Fig.1) was synthesized in Pharmaceutical ResearchInstitute (Warsaw, Poland) as described bySzczepek et al. (23), N-desmethyl imatinib (CASNo. 404844-02-6, Fig. 1) was supplied by TorontoResearch Chemicals Inc. (North York, Canada) andpropranolol hydrochloride ñ the internal standard(CAS No. 318-98-9, Fig. 1) was purchased fromSigma-Aldrich (St. Louis, MO, USA).

Acetonitrile, tert-butyl-methyl ether (bothPOCH, Gliwice, Poland), and methanol (J.T. Baker,Phillipsburg, NJ, USA) were of HPLC grade.Orthophosphoric acid 85% and sodium carbonate

(both Chempur, Piekary ålπskie, Poland),dichloromethane and potassium dihydrogen phos-phate (both POCH, Gliwice, Poland) were of analyt-ical grade. Water was purified by a Milli-Q gradientsystem (Merck Millipore, Billerica, MA, USA).Blank plasma was obtained from the RegionalCenter of Blood Donation and Treatment (Warsaw,Poland). Sodium citrate was used as the anticoagu-lant.

Instrumentation and chromatographic conditions

The HPLC system consisted of a system con-troller SLC-10AVP, a pump LC-10ADVP, anautosampler SIL-10A, a detector SPD 10-AVP, acolumn oven and the data processing softwareClass-VP version 6.13 (Shimadzu, Kyoto, Japan). ASymmetryÆ C18 chromatographic column (250 ◊4.6 mm, 5 µm, Waters, Milford, MA, USA) was pro-tected by a Security Guard C18 (4 ◊ 3 mm,Phenomenex, Torrance, CA, USA). The columntemperature was set at 35 ± 2OC. The mobile phaseconsisting of a phosphate buffer (pH 3) and acetoni-trile (73 : 27, v/v) was delivered at the flow rate of1.2 mL/min. Imatinib and N-desmethyl imatinibwere observed at the wavelength of 265 nm and theon-line wavelength switching to 298 nm was set at4.7 min to record the peak of the internal standard(Fig. 1). The sample volume of 50 µL was injectedonto the column. The analysis time was 7 min.

Stock and working solutions

The stock solutions of imatinib and the internalstandard (both 1 mg/mL) were prepared in methanoland stored in a freezer. The stock solution of N-desmethyl imatinib (0.1 mg/mL) was prepared indichloromethane ex tempore. The working solutions

Figure 1. Chromatogram of the sample containing N-desmethyl imatinib at 250 ng/mL (NDI 3.2 min), imatinib at 3000 ng/mL (IMT 3.6min) and propranolol at 10 µg/mL (IS 5.8 min); with structures and with on-line wavelength switching from 265 nm to 298 nm at 4.7 min

HPLC-UV assay of imatinib in human plasma optimized for... 1497

of imatinib and the internal standard (in 50%methanol, v/v) as well as N-desmethyl imatinib (inmethanol) were stored in a refrigerator. Separatestock solutions were used to obtain spiking solutionsfor the calibration standards and quality control(QC) samples.

Calibration standards and quality control sam-

ples

Calibration standards and QC samples wereprepared by mixing blank plasma with the spikingsolution of imatinib in the ratio of 9 : 1 (v/v). Thecalibration standards were prepared ex tempore atthe following levels: 40, 120, 600, 1200, 1800,2400, 3200 and 4000 ng/mL. The QC samples (120,1800 and 3200 ng/mL) were prepared in larger vol-ume and stored at -20OC together with the studysamples from the volunteers.

Plasma sample preparation

Twenty five µL of the internal standard (80µg/mL) and 50 µL of 2 M sodium carbonate wereadded to 250 µL aliquot of human plasma andmixed. Then, 1.5 mL of tert-butyl-methyl ether wasadded and mixed. Following centrifugation theaqueous phase was frozen, the organic layer wastransferred to a glass tube and evaporated under thestream of nitrogen. The dry residue was reconstitut-ed in 200 µL of 50% methanol.

Validation methodology

The validation was performed according to theregulatory guidelines (24, 25) in compliance withthe principles of Good Laboratory Practice. A nor-mal distribution of measurements was assumed forthe calculation of the precision, accuracy and cali-bration curve parameters. The statistical analysis ofthe stability included the comparison of two sets ofexperimental data assuming a log-normal distribu-tion of the measurements results. It was based on theapplication of confidence intervals (26).

Interferences from the endogenous compoundswere assessed by analyzing blank plasma from sixdifferent sources, including hemolyzed and hyper-lipidemic ones, and twelve pre-dose samples fromboth bioequivalence study phases.

The back-conversion of the metabolite into theparent drug was assessed in plasma spiked with N-desmethyl imatinib at the concentration of 250ng/mL. The analyses were performed ex tempore,after storage for 4 h at ambient temperature and afterlong term storage at -20OC.

The linearity was evaluated within the range of40-4000 ng/mL. The ratio of the peak areas of ima-

tinib to the internal standard was plotted against thenominal concentration of imatinib. A linear regres-sion model was used: y = ax + b, where x was thenominal concentration of imatinib, y was the ima-tinib/internal standard peak area ratio, a and b wereslope and intercept, respectively. The curve wasobtained by a weighted linear regression analysiswith the weighting factor selected according to thesum of percentage relative errors (27).

Accuracy and precision were determined with-in-run (intra-run, within one analytical sequence)and between-run (inter-run, within three analyticalsequences) at four levels: 40 ng/mL (LLOQ) and120, 1800 and 3200 ng/mL (QC samples).

The possibility of the samples dilution wasstudied at 6400 ng/mL. The samples were dilutedwith blank human plasma (1 : 1, v/v) prior to pro-cessing. The results were multiplied by the dilutionfactor of 2. The carry-over was assessed by analyz-ing blank human plasma samples immediately afterthe highest concentration calibration standards(4000 ng/mL).

The peak area for the spiked plasma samplesand the reference solutions were compared to deter-mine an absolute extraction recovery at 120, 1800,3200 ng/mL for imatinib and at 10 µg/mL for theinternal standard.

The stability of the QC samples (120 and 3200ng/mL) was evaluated in an autosampler, aftershort-term storage at ambient temperature, afterlong-term storage at -20OC and after three freeze andthaw cycles. Imatinib and the internal standard sta-bilities in the solutions were assessed during storagein a freezer or in a refrigerator as well as during stor-age for 24 h at ambient temperature.

Reproducibility of the method was evaluatedby reanalyzing the study samples from the volun-teers in separate runs on different days. Calculationswere based on the formula:

% difference = (repeat value ñ initial value)/mean value ◊ 100

The % difference should not be greater than20% of their mean for at least 67% of the repeats.

RESULTS

Method optimization

In order to obtain a good separation of N-desmethyl imatinib, imatinib and the internal stan-dard, chromatographic conditions ñ particularly thetype of the column and the composition of themobile phase ñ were optimized in several steps. Theinitial attempts based on the previously reportedmobile phase consisting of the phosphate buffer and


acetonitrile in the ratio of around 7 : 3 (v/v) (10, 12,15, 16, 22). The final ratio was adjusted to a 250 mmlong chromatographic column, likewise by Roth etal. (16) and Miura et al. (21), which assured goodseparation in the plasma samples. The retentiontimes of the substances in the selected conditionswere 3.2 min, 3.7 min and 5.8 min, respectively. Thewavelength of 265 nm was selected as optimal forthe determination of imatinib, but it was unsuitablefor the internal standard. Therefore, on-line wave-length switching from 265 to 298 nm was appliedduring the analysis (Fig. 2). The use of propranololhydrochloride as the internal standard ñ for the firsttime for imatinib determination ñ allowed to achievethe analysis time of 7 min.

In the study, sample pretreatment was based onthe extraction with tert-butyl-methyl ether. In con-trast to the method by Oostendorp et al. (10) a sodi-um carbonate solution was added to improve theextraction recovery, which allowed to reduce theplasma volume from 1000 µL to 250 µL.

Parrillo-Campigila et al. (7) were the onlyresearchers who successfully applied their HPLC-UV method in a bioequivalence study. Bioequi-valence studies require that at least 80% of theextrapolated area under the concentration time curveAUC(0-∞) should be covered by the measured AUC(0-t)

(8). To meet this criterion, the linearity range of 40-4000 ng/mL was chosen in contrast to Parrillo-Campigila et al. (7) who used the range of 20-3000ng/mL.

Selectivity No peaks were found near the retention times

of imatinib or the internal standard in all of the blank

plasma samples including hemolyzed and hyperlipi-demic ones. The back-conversion of N-desmethylimatinib was not observed immediately after prepa-ration, after storage for 4 h at ambient temperaturenor after storage at -20OC for 239 days (Fig. 2).

Linearity The calibration curve was linear within the

range of 40-4000 ng/mL. For each LLOQ a signal tonoise ratio over 5 was observed. The weighting fac-tor of 1/y2 for the peak area was selected. All regres-sion parameters were statistically significant (signif-icance level α = 0.05, df = n ñ 2). All calibrationcurves (14 from validation and 18 from the study)met the acceptance criteria and none of the calibra-tion standards was rejected (Table 1, Fig. 3). Figure3 shows percentage relative error plotted against theimatinib nominal concentration for all calibrationstandards from the validation (dots in the Figure)and from the study (lines in the Figure). All of themwere ±15% of the nominal value.

Accuracy and precisionThe LLOQ and QCs were quantified with an

acceptable accuracy and precision (Table 2): 90%confidence intervals (CIs) for the percentage of thenominal value were within 80-120% and 85-115%,respectively; 90% CIs for coefficient of variation(CV) did not exceed 20% or 15%, respectively.

Dilution integrity and carry-overThe possibility of a twofold sample dilution was

confirmed for the concentration of 6400 ng/mL. Theaccuracy and precision were 106.9% (90% CI for thepercentage of the nominal value: 105.2-108.6%) and

Figure 2. HPLC chromatograms of the extracts containing N-desmethyl imatinib at 250.0 ng/mL extracted immediately after preparation(A), after being stored for 4 h at ambient temperature (B), after being stored at -20OC (C) for 239 days and extracts containing imatinib(IMT 3.7 min) at LLOQ and propranolol at 10 µg/mL (IS 5.8 min) (D)


1.95% (90% CI for CV: 1.53-3.66%),respectively. The carry-over experi-ment showed the lack of any signalsinfluencing the quantification.

Extraction recoveryThe absolute extraction recovery

of imatinib from human plasma wasconsistent and ranged from 75.6 to81.2%. The absolute extraction recov-ery of the internal standard rangedfrom 84.1 to 87.5% and was unim-paired by the imatinib concentration.

Stability

For each plasma concentrationlevel a 90% CI for the mean stabilityfell within the range of 85-115%(Table 3). The tests confirmed the sta-bility of imatinib and the internal stan-dard in the stock solutions stored for24 and 22 days at = -6OC, respective-ly, as well as in the working solutionsstored for 19 and 40 days at = 12OC,respectively. All solutions were stablefor 24 h at ambient temperature.

Clinical application and incurred

samples reanalysis

The method was successfullyapplied to quantify over 1300 plasmasamples during the bioequivalencestudy of imiatinib mesylate 400 mg.All analytical sequences were accept-ed and none of the calibration stan-dards was rejected. The accuracy andprecision of the calibration standardsachieved during the study were com-parable to the results recorded duringvalidation (Table 1). The results forthe LLOQ and QCs from the within-run validation, between-run validationand from the study confirmed theaccuracy and precision of the method(Table 2). Imatinib concentrationsabove the upper limit of quantifica-tion were not observed, whichenabled to avoid the time-consumingdilution of the samples and proved aproper linearity range. In all pharma-cokinetic profiles the AUC(0-t) coveredat least 80% of the AUC(0-∞), whichconfirmed an appropriate sensitivityof the method (25).

Tab

le 1

. Acc

urac

y (%

of

nom

inal

val

ue)

and

prec

isio

n (C

V)

resu

lts o

f ca

libra

tion

stan

dard

s.

Imat

inib

co

ncen

trat

ion

4012

060

012

0018

0024

0032

0040

00[n

g/m

L)

Val

idat

ion

(n =

14)

Mea

n 40

.811

1.8

587.

012

01.4

1833

.624

48.5

3300

.941

65.3

SD0.

524.

7814

.748

.253

.394

.692

.120

1.5

Acc

urac

y10

1.9

93.2

97.8

100.

110

1.9

102.

010

3.2

104.

1

90%

CIs

101.

3-10

2.5

91.3

-95.

096

.7-9

9.0

98.2

-102

.010

0.5-

103.

310

0.2-

103.

910

1.8-

104.

510

1.7-

106.

5

Prec

isio

n1.

284.

272.

514.

012.

913.

862.

794.

84

90%

CIs

1.06

-1.7

73.

22--

5.41

1.99

-3.3

43.

26-5

.46

2.40

-4.0

23.

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Over 100 samples were selected for theincurred samples reanalysis to illustrate themethodís reliability. The incurred samples werederived from different volunteers, close to the max-imum concentration and in the elimination phase.For 69% of the results the % difference was notgreater than 20%, which indirectly confirmed thatthe metabolites ñ especially N-desmethyl imatinib ñdo not interfere with the imatinib quantification(25).

DISCUSSION AND CONCLUSION

The method was optimized for a bioequiva-lence study. Important innovations included: a shortanalysis time in comparison to other HPLC-UVmethods, the selection of propranolol hydrochlorideas the internal standard and the linearity range dedi-cated for a single 400 mg dose as well as an opti-mized liquid-liquid extraction procedure. The vol-ume of the extrahent and the plasma volume in com-parison to the previous method with tert-butyl-methyl ether was reduced (10), thus making thesample volume comparable to other extractions(17). A lower sample volume is an important advan-tage of a bioanalytical method that is convenient forthe study subjects and facilitates the blood samplingprocedure in clinical sites. Although the recoveriesof several methods (7, 11, 16, 20) were higher com-pared to the presented method (above 90%), the

recovery at the level of around 80% was sufficientfor the intended purpose.

The use of propranolol hydrochloride allowedto reduce the analysis time to 7 min, but it has alsolimited the use of the method to the samples takenfrom the subjects who were not treated with propra-nolol like healthy volunteers in bioequivalence stud-ies. Only two methods had shorter analysis time: 4min (14) and 5 min (13), respectively. In both meth-ods and in 7-min method (22) the sensitivity wasunsuitable to study imatinib pharmacokinetics pro-file following a single administration of 400 mg.Birch et al. (14) demonstrated the calculated valueof LLOQ at 2 ng/mL, but it was outside the studiedcalibration range - 50-10000 ng/mL - which made ituseless for bioequivalence studies.

Most of the HPLC-UV methods for the imatinibquantification in human plasma were used for theroutine monitoring of imatinib and N-desmethyl ima-tinib in patients (10-12, 15, 16, 21-23) what requiredwider linearity range. In the case of imatinib the meanvalues of the maximum plasma concentration rangefrom about 1600 to 3000 ng/mL after a single 400 mgdose administration (1, 5-7). The steady-state plasmaconcentrations in patients - ranging from 1093 ng/mLto 4980 ng/mL (17) - are useless for planning studiesfollowing a single dose in healthy subjects. Someauthors do not explain the extended calibrationranges, e.g., linearity range of 25-25000 ng/mL vs.reported the maximum concentration of only 1385

Figure 3. Calibration standards ñ percentage relative error plotted against the imatinib nominal concentration 40, 120, 600, 1200, 1800,2400, 3200 and 4000 ng/mL; dots in figure ñ result from the validation (n = 14), lines in figure ñ result from the study (n = 18); weight-ing factor w = 1/y2


ng/mL (15). In contrast, Tan et al. (12) determined themean maximum concentration in patients at 3415ng/mL using linearity ranged of 50-1800 ng/mL. Itrequired the dilution of numerous samples whatextended the sample preparation process - a clear dis-advantage in a study with a large number of samples.

The lowest sensitivity of the HPLC-UV meth-ods, defined as LLOQs, was 10 ng/mL (9, 10, 21).Birch et al. (14) demonstrated the LLOQ at 2 ng/mLbut it was outside their calibration range. In turn,Pirro et al. (23) reported the linearity range of 5-10000 ng/mL but the calculated LLOQ was only 50ng/mL. The sensitivity at 10 ng/mL is unnecessaryto study pharmacokinetic profiles following a single400 mg imatinib dose. We chose the LLOQ of 40ng/mL because, especially in the UV analysis ofplasma samples, lower values may lead to problemswith the methodís selectivity, e.g., the integration ofpeaks or high signal to noise ratio. However, thethree mentioned methods with the LLOQ at 10

ng/mL were applied to measure imatinib in clinicalsamples and the LLOQ did not limit their use in abioequivalence study. The most sensitive methodswere limited by a long chromatographic run time, 50min (10), 40 min (9) and 30 min (21), respectively.The analysis of 1300 samples takes about 6 dayswith the presented method and 45, 36 and 27 daysusing the other methods, respectively. In turn, themethod by Velpandian et al. (15), characterized bythe LLOQ at 30 ng/mL and a 10 min analysis time,lacked the selectivity evaluation against N-desmethyl imatinib.

Important task in the bioanalytical methoddevelopment is minimizing the impact of the mainor unstable metabolites on the determination of theparent drug (28, 29). In most of the HPLC-UVmethods imatinib and N-desmethyl imatinib werechromatographically separated (9-12, 15, 16). Inthis study, the particular attention was paid to themetabolite back-conversion during the storage and

Table 2. Accuracy (% of nominal value) and precision (CV) results of LLOQ and QCs.

Sample type LLOQ QC QC QC

Imatinib concentration 40 120 1800 3200

[ng/mL)

Validation: within-run (n = 6)

Accuracys 97.5 100.2 96.5 103.490% CI 94.1-100.9 96.9-103.5 94.2-98.8 100.7-106.1

Precision 4.26 4.02 2.85 3.183.1890% CIs 3.14-7.51 2.96-7.08 2.10-5.02 2.34-5.60

Validation: between-run (n = 18)

Accuracys 102.5 98.3 97.9 103.590% CI 100.1-105.0 94. -9101.7 96.6-99.1 101.5-105.5

Precision 5.88 8.40 3.07 4.7490% CIs 4.87-7.63 6.96-10.9 2.55-3.99 3.93-6.16

During the study

n 18 27 29 29Accuracy 100.6 102.0 104.1 101.590% CIs 99.9-101.3 99.9-104.1 102.1-106.1 99.4-103.6Precision 1.71 6.27 6.00 6.4690% CIs 1.43-2.24 5.47-7.84 5.37-7.59 5.64-7.98

Table 3. Stability of imatinib in plasma expressed in % as 90% CI (n = 6).

Stability Autosampler Freeze-thaw Short-term Long-term

Temperature Ambient -20OC Ambient -20OC

Storage period 66 h 3 cycles 4 h 52 days

Imatinib 120 100.1-102.0 88.5-95.8 97.6-108.5 100.3-104.8concentration

[ng/mL) 3200 101.6-102.8 99.1-105.6 97.6-101.9 102.7-109.5


processing of the plasma samples. Prior to theanalysis the real samples are frozen at the clinicalsite, transported to a bioanalytical laboratory,thawed (sometimes two or three times) andprocessed at ambient temperature. The metabolitesmight back-convert during those steps. So, theback-conversion tests were performed: ex tempore,short-term (4 h at ambient temperature) and long-term (239 days in a freezer). The plasma concentra-tion of the metabolite (250 ng/mL) was selectedbased on the expected maximum plasma concentra-tion of N-desmethyl imatinib after the oral adminis-tration of a 400 mg dose of imatinib (4, 5). The testsdid not indicate any back-conversion of N-desmethyl imatinib (Fig. 2). Admittedly, Davies etal. (11) suggested performing stability tests awayfrom sunlight, but imatinib and its main metaboliteare insensitive to daylight in contrast to sunitinib(30).

The methodís advantages include: (a) shortanalysis time of 7 min, (b) simple sample prepara-tion based on the liquid-liquid extraction and (c)lack of the influence of the main metabolite N-desmethyl imatinib. It makes the method a valuablealternative to the existing HPLC-UV methods forthe routine analysis of large numbers of clinicalsamples. For bioequivalence studies N-desmethylimatinib does not need to be quantified, therefore theback-conversion before analysis was studied. Thelinearity range of 40-4000 ng/mL was optimized toevaluate imatinib pharmacokinetics following a 400mg single oral dose of imatinib mesylate. Themethodís reliability was confirmed by its successfulapplication in the bioequivalence study.

Acknowledgments

The work was supported by the EuropeanUnion (European Regional Development Fund)under the Innovative Economy OperationalProgramme 2007-2013 (POIG.01.03.01-14-032/12-01). The authors gratefully acknowledge ProfessorAndrzej Kutner for his critical review of the manu-script and Ms. Krystyna Serafin-Byczak for hertechnical assistance in the development of the pre-sented method.

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3. Gschwind H.P., Pfaar U., Waldmeier F.,Zollinger M., Sayer C. et al.: Drug Metab.Dispos. 33, 1503 (2005).

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7. Guideline on the Investigation of Bioequi-valence. Committee for Medicinal Products forHuman Use (CPMP/EWP/QWP/1401/98/ Rev.1 Corr.**). London 2010.

8. Schleyer E., Pursche S., Kohne C.H., Schuler U.,Renner U. et al.: J. Chromatogr. B 799, 23 (2004).

9. Oostendorp R.L., Beijnen J.H., SchellensJ.H.M., van Tellingen O.: Biomed. Chromatogr.21, 747 (2007).

10. Davies A., Hayes A.K., Knight K., WatmoughS.J., Pirmohamed M., Clark R.E.: LeukemiaRes. 34, 702 (2010).

11. Tan K.L., Ankathil R., Gan S.H.: J.Chromatogr. B 879, 3583 (2011).

12. Golabchifar A.A., Rouini M.R., Shafaghi B.,Rezaee S., Foroumadi A., Khoshayand M.R.:Talanta 85, 2320 (2011).

13. Birch M., Morgan P.E., Handley S., Ho A.,Ireland R., Flanagan R.J.: Biomed. Chromatogr.27, 335 (2013).

14. Velpandian T., Mathur R., Agarwal N.K., AroraB., Kumarc L., Gupta S.K.: J. Chromatogr. B804, 431 (2004).

15. Roth O., Spreux-Varoquaux O., Bouchet S.,Rousselot P., Castaigne S. et al.: Clin. Chim.Acta 411, 140 (2010).

16. Miura M., Takahashi N.: Drug Metab.Pharmacokinet .31, 12 (2016).

17. Bakhtiar R., Khemani L., Hayes M., Bedman T.,Tse F.: J. Pharm. Biomed. Anal. 28, 1183 (2016).

18. Jung J.A., Kim N., Yang J.-S., Kim T., Kim J.-R. et al.: Drug Res. 64, 651 (2014).

19. Widmer N., BÈguin A., Rochat B., Buclin T.,Kovacsovics T. et al.: J. Chromatogr. B 803,285 (2004).

20. Miura M., Takahashi N., Sawada K.: J.Chromatogr. Sci. 49, 412 (2011).

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22. Pirro E., De Francia S., De Martino F., Fava C.,Ulisciani S. et al.: J. Chromatogr. Sci. 49, 753(2011).


23. Szczepek W.J., KosmaciÒska B., BielejewskaA., £uniewski W., SkarøyÒski M., Rozmary-nowska D.: J. Pharm. Biomed. Anal. 43, 1682(2007).

24. Guideline on bioanalytical method validation.European Medicines Agency (EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2**). London2011.

25. Guidance for Industry: Bioanalytical methodvalidation. U.S. Department of Health andHuman Services. Food and Drug Adminis-tration. Center for Drug Evaluation andResearch (CDER) and Center for VeterinaryMedicine (CVM). 2001.

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Received: 27. 07. 2016


Reactive oxygen species (ROS) are formed inoxidation of various cell constituents as DNA, lipidsand proteins and consequently cause oxidative dam-age of cellular substance leading to cell death (1).The oxidative damage of DNA induced by ROS leadto certain cancers, and ROS may also play a role incell cycle progression. ROS are implicated innumerous pathological events including metabolicdisorders, cellular aging, reperfusion damage ofDNA, inflammation and atherosclerosis (2). In thatlight, searching for new, antioxidant agents seems tobe well grounded (3). It was demonstrated thatoxidative stress and reactive oxygen species produc-tion are involved also in epilepsy pathogenesis. Thexanthone nucleus comprises an important class ofoxygenated heterocycles. They are found in naturalproducts, especially in higher plants, lichens andfungi. Plants belonging to the Guttiferae family arethe richest in xanthone derivatives. The most popu-lar is Garcinia mangostana, a tropical evergreentree. Its origin is in Southeast Asia. It can now befound in Northern Australia, Brazil, CentralAmerica, Hawaii, Southern India, Indonesia,Malaysia, Thailand, and other tropical countries.The edible fruit is deep reddish purple when ripe. In

Asia, it is known as the ìQueen of Fruitsî due to itspleasant flavor (4). The fruitís hull of α-, β- and γ-mangosteen, 3-isomangosteen, gartanin, 8-desoxy-gartanin has been used for hundreds of years inSoutheast Asia as a medicine for skin infections,wounds, dysentery and diarrhoea, but those biologi-cal benefits were not related with the xanthonestructure (5). Nowadays, mangosteen is used as aningredient in several popular commercially availablenutritional supplements with antioxidant, cardiovas-cular, immunestimulating activity, includingVemma (Vemma Co.) and Xango (Xango Co.) (6).It is known from the literature that xanthone deriva-tives are characterized with diverse biological activ-ities including tuberculostatic (7), antimicrobial (8),cardiovascular (9), antiinflamatory (10) and antioxi-dant (11, 12). Taking into account antioxidativeproperties, compounds can act as metal chelators,free radical scavengers, as well as inhibitors of lipidperoxidation (13). There are evidences that oxida-tive stress can be involved in seizure generation (14,15). What is more, beneficial effects of antioxidantsin animal models of epileptic seizures was observed(16), therefore, antioxidant properties could be rec-ommended for newly synthesized antiepileptic com-

DRUG BIOCHEMISTRY

ANTIOXIDANT ACTIVITY OF XANTHONE DERIVATIVES

RENATA FRANCIK*, NATALIA SZKARADEK, DOROTA ØELASZCZYK and HENRYK MARONA

Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy,Jagiellonian University, Medical College, Medyczna 9, 30-688 KrakÛw, Poland

Abstract: Certain xanthone derivatives, such as these present in mangosteen fruits, show strong antioxidantactivity. On the other hand, evidences accumulated that oxidative stress is involved in epileptogenesis.Therefore, the aim of the present study was to estimate total antioxidant capacity (expressed as a ferric reduc-ing antioxidant power - FRAP) and evaluate ability to scavenge free radicals (DPPH methods) by xanthonederivatives showing antiepileptic activity. Selected 2-(aminomethyl)-9H-xanthen-9-one derivatives sharedstructural features, such as chlorine substituent in xanthone ring and different chiral (or not) alkanol groups atthe nitrogen atom. The results of antioxidant activities among racemates revealed the highest activity for com-pound (R/S)-3 (31.7% in diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and (0.184 ± 0.003 mM Fe2+/L)in FRAP assay. Among tested pair of enantiomers we observed that (R)-1 and (R)-2 showed higher reductioncapacity ((R)ñ1: 0.096 ± 0.007 mM Fe2+/L; (R)-2: 0.048 ± 0.005 mM Fe2+/L, respectively) and stronger DPPHscavenging activity ((R)-1: 31 ± 3.0%; (R)-2: 29 ± 2.5%, respectively) comparing to their (S)-enantiomers andracemates.

Keywords: xanthone derivatives, oxidative stress, DPPH test, FRAP

1505

* Corresponding author: e-mail: [email protected]; phone: +48126205507, fax: +48126205405

1506 RENATA FRANCIK et al.

pounds. New derivatives of xanthone are the subjectof the research carried out in our Department ofBioorganic Chemistry. Anticonvulsant activity andneurotoxicity were evaluated under theAnticonvulsant Screening Program (ASP) at theNational Institute of Neurological Disorders andStroke, National Institutes of Health, Bethesda,USA.

Most of the structures mentioned below provedtheir beneficial properties and were previouslydescribed (17-20). Compounds 2 R,S; 2 R and 2 Sdisplayed significant anti-MES (maximal elec-

troshock) activity in mice with protective index(TD50/ED50) of 5.84, 6.23 and 6.85, corresponding tothat of phenytoin, carbamazepine and valproate.Moreover, compound 2 S revealed low micromolaraffinity to the voltage-dependent Ca2+ channels,comparable to that of carbamazepine (17, 18).Xanthone derivatives from the first series (1 S, 1 R,1 R,S) were not so effective and acted in higherdoses than second series (2 R,S; 2 R and 2 S) (18,19).

Compound 3 R,S with chlorine atom at posi-tion 7 in the xanthone scaffold was active in MES

Table 1. Xanthone derivatives containing chlorine in the aromatic ring and different alkyl substituent at the nitrogen atom.

Compound Structure and IUPAC name

(S)-1

(S)-6-chloro-2-{[(1-hydroxypropan-2-yl)(methyl)amino]methyl}-9H-xanthen-9-one hydrochloride

(R)-1

(R)-6-chloro-2-{[(1-hydroxypropan-2-yl)(methyl)amino]methyl}-9H-xanthen-9-one hydrochloride

(R,S)-1

(R,S)-6-chloro-2-{[(1-hydroxypropan-2-yl)(methyl)amino]methyl}-9H-xanthen-9-one hydrochloride

(S)-2

(S)-6-chloro-2-{[(1-hydroxypropan-2-yl)amino]methyl}-9H-xanthen-9-one

(R)-2

(R)-6-chloro-2-{[(1-hydroxypropan-2-yl)amino]methyl}-9H-xanthen-9-one

(R,S)-2

(R,S)-6-chloro-2-{[(1-hydroxypropan-2-yl)amino]methyl}-9H-xanthen-9-one

(R,S)-3

(R,S)-2-chloro-7-{[(1-hydroxybutan-2-yl)amino]methyl}-9H-xanthen-9-one

4

6-Chloro-2-{[ethyl(2-hydroxyethyl)amino]methyl}-9H-xanthen-9-one hydrochloride

Antioxidant activity of xanthone derivatives 1507

test in the dose of 30 mg/kg in 87% of the examinedmice (20).

Herein we report on radical scavenging andantioxidant activities of the synthesized xanthonederivatives using 2,2-diphenyl-1-picrylhydrazyl(DPPH) and ferric reducing antioxidant power(FRAP) assays.


The reagents such as vitamin C, quercetin,trolox, 2,2-diphenyl-1-picrylhydrazyl and 2,4,6-tri-(2-pyridyl)-s-triazine were of analytical grade andwere obtained from Sigma-Aldrich ChemicalCompany (Steinheim, Germany). All other reagents(FeSO4, FeCl3 and methanol) were purchased fromAvantor Performance Materials Poland S.A.

Xanthone derivatives: (R/S)-1; (S)-1; (R)-1;(R/S)-2; (S)-2; (R)-2; (R/S)-3 and 4 (Table 1) weresynthesized at the Department of BioorganicChemistry, UJ CM, KrakÛw, Poland. Their struc-tures were confirmed by 1H NMR and IR spectra(17-20). Enantiomers were checked for their purityby measuring their specific rotation (19, 20). For thestudies, compounds were dissolved in water usingultrasounds and tested in the following concentra-tions: 0.3, 0.6, 1, 50 and 200 µM. Reference antiox-idants were used in the same concentrations as test-ed compounds: L-ascorbic acid (vit. C, dissolved inwater), trolox (dissolved in methanol) and quercetin(polyphenol dissolved in methanol). Spectrophoto-meter Cecil CE 7200 BioAquarius (Cecil Instru-ments Limited, Cambridge, UK) was applied for themeasurement of absorbance. Assays were carriedout in triplicate. Results are given as the means withstandard deviation.

DPPH free radical scavenging activity assay

Antioxidant properties of the investigatedgroup of compounds were measured by DPPH testbased on the method reported by Blois (21). In gen-

eral, DPPH in its stable radical form absorbs at 517nm, but upon reduction by an antioxidant present ina sample its absorption decreases. Solution (0.6mM) of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) wasprepared in methanol. Fixed volume (25 µL) of solu-tions of each compound or reference substance(0.3ñ200 µM) were added to 1 mL of DPPH.Solutions were mixed and incubated in dark at roomtemperature for 30 min. After that time, theabsorbance of samples were measured at 517 nm(As) against blank sample (methanol) (Ab). As acontrol, absorbance of DPPH solution with 25 µL ofdistilled water was measured (Ac). The capability oftested compounds to scavenge the DPPH radical(antioxidant activity) was calculated using the fol-lowing equation:

Ac ñ AsDPPH [%] = ññññññññññññ × 100Ac ñ Ab

where Ac was the absorbance of the control, As ofthe sample and Ab of the blank (methanol).

Measurement of the total antioxidant capacity

The FRAP assay, which is the modification ofBenzie and Streinís method (22), was applied tomeasure the ability of xanthone derivatives toreduce Fe3+ to Fe2+ ions in acidic environment (pH3.6). Fe2+ ions in the presence of 2,4,6-tripyridyl-S-triazine (TPTZ) forms Fe2+-TPTZ intensive bluecomplexes, with maximum absorbance at 593 nm.Reaction mixture consisted of acetate buffer 0.3 M,(pH 3.6); TPTZ 0.01 M and iron(III) chloride 0.02M. Samples were prepared by adding 50 µL of test-ed compound or standard in different concentrations(0.3ñ200 µM) to 1 mL of reaction mixture. Aftermixing, samples were incubated at 37OC for 30 min.After that time, the absorbance was measured at awavelength of λ = 593 nm. Blank test was per-formed similarly, but to a reaction mixture 50 µL ofdistilled water was added. The antioxidant activitywas expressed in micromoles of ferrous ions perliter produced by tested compound or standard,

Figure 1. DPPH [%] concentration values for the standard and xanthone derivatives of the series 1, 2 and substances 3R,S and 4

1508 RENATA FRANCIK et al.

which was calculated from the calibration curve ofiron(II) sulfate (100 to 1000 µM/L of FeSO4).

RESULTS AND CONCLUSIONS

Number of reports concerning studies onplant extracts from Garcinia rigida, Garcinia man-gostana, Cudrania tricuspidata or Cratoxylumcochinchinense demonstrated their various biolog-ical activities, such as anti-inflammatory, antibac-terial, antifungal, antioxidant, cytotoxic and anti-HIV (23-26). Plants mentioned above are source ofxanthone derivatives, that were isolated and identi-fied as (poly)phenols, possessing one or morehydroxyl groups in the aromatic scaffold (27, 28).Among synthetic xanthone derivatives, we report-ed some compouds with promising anticonvulsantproperties being derivatives of 6- or 7-chloro-2-(aminomethyl)-9H-xanthen-9-one. For this study,we selected derivatives with defined anticonvul-sant activity possessing hydroxyl group in the sidealkyl chain (Table 1). Their ability to ìsweep offîfree radicals was measured and expressed as a per-centage of activity (DPPH test - Fig. 1). In case ofthe FRAP method, production of Fe2+ in the pres-ence of xanthone derivatives was calculated(FRAP test - Fig. 2).

Figure 1 illustrates a decrease in the concentra-tion of DPPH due to the scavenging ability of xan-thone derivatives and standards. The scavengingeffect decreased in the order of quercetin > vitaminC > (R/S)-3 > (R)-1 > (R)-2 > trolox > 4 > (S)-1 >

(R/S)-1 > (S)-2 > (R/S)-2 yielding 63.5, 45.4, 31.7,31.4, 27.6, 27.3, 26.6, 25.6, 20.6 and 15.6%, at theconcentration of 200 µM, respectively. Changingconcentrations of tested compounds from 200 to 50µM resulted in greatest difference in outcomes forall substances. In case of (R/S)-2, a decrease in scav-enging activity was 67%, while for vit. C was only32%. At the lowest tested concentration (0.3 µM)

the calculated scavenging activity for racemic (R/S)-

2 was of negative value, what can suggest pro-oxidative properties of racemic compound. Sucheffect was not observed in case of both of its enan-tiomers (S)-2 and (R)-2. Comparing scavengingeffect of pairs of enantiomers and racemates (comp.1 and 2) it can be readily observed that R-enan-tiomers are more potent free radicals scavengers(Fig. 1). We have previously reported some differ-ences in free radical scavenging activity betweenstereoisomers of β-carboline derivatives (29).

In case of FRAP assay, the highest Fe2+ contentin the tested samples was observed for compounds(R)-1 and (R/S)-3 (Fig. 2). The reducing power ofthese compounds was higher than all three referencesubstances tested. Only at the highest concentration(200 µM), vitamin C showed stronger antioxidativeactivity than compound (R)-1, while quercetin atconcentration 1 µM more effectively reduced Fe3+

than (R/S)-3. Concerning chirality of xanthonederivatives, both R-enantiomers ((R)-1 and (R)-2)showed the tendency to increase value of FRAPwith the decrease of concentration. In case of stan-dards, only vit. C decreased FRAP value when dilut-ing samples, thus showing reducing properties. Forcompound (S)-1, FRAP values did not significantlydiffer in all of the tested concentrations. Compound(R/S)-2 was the least active in the studied group ofxanthone derivatives. Generally (R)-enantiomersshowed stronger ferric reducing antioxidant powerthan their (S)-enantiomers and racemates.Additionaly, (R/S)-1 showed prooxidative proper-ties at the lowest tested concentration (0.3 µM),what overlapped with the observation in DPPHassay.

When comparing antioxidant activity of testedcompounds with their antiepileptic properties andstructure, most active in central nervous systemcompound (S)-2 (17, 18) with chlorine atom in theposition of 6 and propanol as alkanol substituent,

Figure 2. FRAP [mM Fe2+/L] concentration values for the standard and investigated substances of the series 1, 2 and substances 3R,S and 4

Antioxidant activity of xanthone derivatives 1509

possessed only very mild antioxidant properties.Racemic (R/S)-3, bearing chlorine in position 7 ofxanthone scaffold and butanol substituent at nitro-gen atom, showed moderate anticonvulsant protec-tion (19) and revealed the highest antioxidant effectin tested series of compounds. On the other hand,(R)-1, with chlorine substituent in position 6 of xan-thone structure being tertiary amine with propanolsubstituent and methyl group, exhibited the highestferric reduction power but in preliminary anticon-vulsant screening was less effective than other com-pounds (17, 18).

In conclusion, the obtained results of antioxi-dant studies for compounds affecting CNS functionsin the group of 2-(aminomethyl)-9H-xanthen-9-onederivatives with alkanol substituent at the nitrogenentitled to lead further studies to search for newantiepileptic structure with good antioxidant proper-ties. It requires of course more advanced methodol-ogy.

Acknowledgments

This work was supported by theK/ZDS/005487 and K/DSC/001962 programs.

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Received: 30. 10. 2015


Yeasts are always the most frequent organisminvolved in invasive fungal infections. C. albicans isthe fourth most common pathogen among microor-ganisms isolated in intensive care units (afterStaphylococcus aureus, Enterococcus and Gram-negative rods). An increase in infections due to nonalbicans species of Candida has been observed inthe recent years (1, 2). Fungal infections are a lead-ing cause of mortality in patients with chemothera-py, neutropenia and HIV (3). The discovery of theazole antifungal compounds, ketoconazole, itra-conazole, and fluconazole, allowed for a broaderspectrum of antifungal treatment and a shorter treat-ment duration (4). These drugs act by inhibitingcytochrome P450-dependent ergosterol synthesisand cytochrome C oxidative and peroxidativeenzymes. This disruption of enzymic processes ulti-

mately leads to fungal cell death. Itraconazole hasactivity against fungi (Aspergillus spp., Sporothrixschenkii) and yeasts (Histoplasma, Blastomyces,and Coccidioides spp.) when compared with keto-conazole (1).

During the last decade, a marked increase inresistance of C. albicans and non- C. albicans toazole and other antifungal treatment has beenobserved (5-8). The search and development of newantifungal agents is expected to offer new opportu-nities for both prophylaxis and treatment of fungalinfections in the immunocomprised host (9).

Series compounds with β-resorcylthiocar-bamoyl moiety from group of thiobenzanilides sub-stituted in the N-aryl ring (10, 11) and N-hetero-cyclic amides (12) were achieved in our laboratory.They show a wide spectrum of antifungal activity in

IN VITRO ANTIFUNGAL ACTIVITY OF 2,4-DIHYDROXY-N-(3-THIOXO-3H-1,2,4-DITHIAZOL-5-YL)BENZENECARBOTHIOAMIDE

CECYLIA £UKASZUK1, ELØBIETA KRAJEWSKA-KU£AK1*, ANDRZEJ NIEWIADOMY2

and BEATA SZYNAKA3

1Department of Integrated Medical Care, Medical University of Bia≥ystok, M. Curie Sk≥odowskiej 7a, 15-096 Bia≥ystok, Poland

2University of Life Sciences in Lublin, Department of Chemistry, Lublin, Poland3Department of Histology and Embryology, Medical University of Bia≥ystok, Poland

Abstract: An increase in infections due to non albicans species of Candida has been observed in the recentyears. The aims of this study were to determine the antifungal activity of 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide (DNTDB) against C. albicans, non-C. albicans, dermatophytes, andmolds and to evaluate the enzymatic activity of C. albicans strains. We used reference strains C. albicans 10231ATCC, 200 C. albicans strains, 100 non-C. albicans, 19 dermatophyte strains, and 21 mold strains isolated fromdifferent ontocenoses from patients. DNTDB revealed a mean minimum inhibitory concentration (MIC) of 12.5µg/mL against the reference C. albicans 10231 ATCC strain on Sabouraud agar (SA) and of 6.5 µg/mL onRoswell Park Memorial Institute (RPMI) medium. The mean MIC for C. albicans isolates was of 22.01 ± 7.5µg/mL on SA, 17.8 ± 7.4 µg/mL on yeast nitrogen base (YNB), and 16.9 ± 7.9 µg/mL on RPMI medium. Themean MIC for non-C. albicans isolates was of 22.4 ± 12.4 µg/mL on SA, 18.2 ± 8.6 µg/mL on YNB and 15.2± 9.03 µg/mL on RPMI. Against Trichophyton mentagrophytes v. granulosum, the mean MIC was 10.9 ± 2.04µg/mL after 5 days of incubation and 21.9 ± 3.8 µg/mL after 15 days, while Trichophyton mentagrophytes v.interdigitale showed a mean MIC of 13.3 ± 5.5 µg/mL and of 20.3 ± 6.1. µg/mL after the same incubation peri-ods, respectively. DNTDB manifested a MIC over the test range of 25ñ100 µg/mL for molds after 5 days ofincubation and inhibited the enzymatic activity of Candida strains. It seems, the new DNTDB demonstratespotential antifungal activity against yeast-like fungus strains, dermatophytes, and molds in vitro.

Keywords: 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide, Candida, molds, der-matophytes, antifungal activity

1511

* Corresponding author: e-mail: [email protected]; phone/fax +48857485528

1512 CECYLIA £UKASZUK et al.

relation to molds (13), yeasts (13), dermatophytes,and strong inhibition action comparable with com-mercial antimycotic drugs. Furthermore, the ben-zothiazoles have also antitumor and antiviral activi-ties in vitro (7).

Taking into account the wide application ofantifungal medicines with azole moiety, 2,4-dihy-droxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benze-necarbothioamide (DNTDB) was produced as acompound with expected antifungal activity.

The aim of this study was to determine the anti-fungal activity of DNTDB against C. albicans, non-C. albicans, dermatophytes, and molds.

EXPERIMENTAL

Chemistry

DNTDB belongs to group of thiobenzanilidessubstituted in the N-aryl ring and N-heterocyclicamides.

One chemical compound - 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbo-thioamide ñ DNTDB (18) having the lowest MICvalue was used for the studies (Fig. 1).

Antifungal activity

In order to define the antifungal activity ofDNTDB we used reference strains C. albicans 10231ATCC, 200 of C. albicans strains, 100 of non-C. albi-cans, 21 dermatophytes strains and 30 molds strainsisolated from different ontocenoses from patients.

The DNTDB was dissolved in 1% DMSO.Susceptibility testing was performed by the agardilution method. For yeasts, dermatophytes andmolds MICs were determined according to ClinicalLaboratory Standard Institute (CLSI) reference doc-ument M27-A3 (6).

The yeasts, dermatophytes and molds were cul-tivated on Sabouraudís medium ñ SA (Bio-Rad,Warsaw, Poland) and the yeasts on RPMI (Sigma,Warsaw, Poland).

Table 1. Hydrolytic enzymes and their substrates assayed using API ZYM test.

No. Enzyme assayed Substrate

I Phosphatase alcaline 2-naphthylphosphate

II Esterase (C4) 2-naphthylbutyrate

III Esterase lipase (C8) 2-naphthylcapylate

IV Lipase (C14) 2-naphthylmyristate

V Leucine arylamidase L-leucyl-2-naphthylamide

VI Valine arylamidase L-leucyl-2-naphthylamide

VII Cystine arylamidase L-cystyl-2-naphthylamide

VIII Trypsin N-benzoyl-DL-arginine-2-naphthylamide

IX Chymotrypsin N-glutaryl-phenylalanine-2-naphthylamide

X Phosphatase acid 2-naphthylphosphate

XI Naphthol-AS-BI-phosphohydrolase Naphthyl-AS-BI-phosphate

XII α-galactosidase 6-Br-2-naphthyl-αD-galactopyranoside

XIII β-galactosidase 2-naphthyl-βD-galactopyranoside

XIV β-glucuronidase Naphthol-AS-BI-βD-glucuronide

XV α-glucosidase 2-naphthylyl-αD-glucopyranoside

XVI β-glucosidase 6-Br-2-naphthyl-βD-glucopyranoside

XVII N-acetyl-β-glucosaminidase 1-naphthyl-N-acetylo-βD-glucosaminide

XVIII α-mannosidase 6-Br-2-naphthyl-αD-mannopyranoside

IX α-fucosidase 2-naphthyl-α-L-fucopyranose

Figure 1. 2,4-Dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)ben-zenecarbothioamide (DNTDB)

In vitro antifungal activity of... 1513

The yeasts were identified to the species levelby the CandiSelect (Bio-Rad, Warsaw, Poland)),Fungiscreen 4H (Bio-Rad, Warsaw, Poland),Auxacolor (Bio-Rad, Warsaw, Poland)) tests.Dermatophytes and molds were identified by stan-dard methods. Prior to antifungal susceptibility test-ing, each isolate was passaged on SA or YNB medi-um and RPMI medium to ensure optimal growthcharacteristics.

Starting inocula were adjusted by the spec-trophotometric method densitometr (BioMerieux,Warsaw, Poland) to 1 ◊ 105 CFU/mL. Concen-trations of the DNTDB were ranging from 0.025 to200 µg/mL. Plates were incubated at 37OC and readafter 24 h incubation. A solvent control was includ-

ed in each set of assays; the DMSO solution at max-imum final concentration of 1% had no effect onfungal growth.

The MICs of the compound against dermato-phytes were determined on SA medium. Concen-trations of the DNTDB were ranged from 0.025 to200 µg/mL.

Dermatophytes inocula were prepared from 3weeks colonies, cultured on SA medium. Controlplates with SA medium without the DNTDB or with1% DMSO were prepared. Microcultures were incu-bated at 27OC, and the MICs values were read after5 and 15 days.

Molds inocula contained 1 ◊ 105 CFU/mL.Petrie disks with tested media and serial dilutions of

Table 2. List of biotypes based on the available literature.

ENZYMES

BIOTYPESE 2

E 6 E 11E 15

E 17ENZYMATIC

Esterase Valine Naphthol-AS-BI-

-glucosidaseN-acetyl-

arylamidase phosphohydrolase glucosaminidase

according to Williamson et al. (22)

A + + + + +

B + - + + +

C + + + - +

D + + - + +

E + + + - -

F + + + + -

G + - + + -

H + + - - -

according to Kurnatowska and Kurnatowski (11)

I - - - - +

J - - - + +

F + + - + -

L + - + - +

M + - + - -

N + - - - +

according to Krajewska-Ku≥ak et al. (9)

O + - - - -

P + - - + -

R - + + + +

according to Krajewska-Ku≥ak et al. (9), and Batura-Gabryel et al. (2)

S + + - - +

T + - - + +

acccording to Brajer et al. (3)

T - + + - -

U - + + - +

W - + - + -


Table 3. MICs of 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide values against the yeast-like fungi strains,dermatophytes, and molds on the different media.

SA YBB RPMIYeast-like fungi medium medium medium

p value

strains MIC(Mean value/Range; µg/mL)

Reference C. albicans 10 231 ATCC strains 25 12.5 6.5 NS

(n = 1)

C. albicans strains 22.01 ± 7.5 17.8 ± 7.4 16.9 ± 7.9 < 0.001 vs.(N = 200) (3-50)* (3ñ25) (6.5ñ25) YNB, RPMI

Non- C. albicans 22.4 ± 12.4 18.2 ± 8.6 15.2 ± 9.0 < 0.01 vs.strains (n = 100) (6.25ñ50) (3ñ25) (3ñ25) YNB, RPMI

C. glabrata strains 18.3 ± 11.2 15.9 ± 8.1 12.2 ± 7.3 NS(n = 30) (6.25ñ50) (3ñ25) (3-25)

C. tropicalis strains 22.5 ± 10.9 21.3 ± 11.3 17.9 ± 12.2 NS(n = 20) (12.5ñ50) (12.5ñ50) (6.5ñ50)

C. krusei strains 19.4 ± 6.2 14.8 ± 7.4 12.1 ± 6.5 NS(n = 20) (12.5ñ25) (3ñ12.5) (6.5ñ25)

Rhodotorula rubra 32.5 ± 15 20 ± 6.1 18.7 ± 6.9 NSstrains (n = 15) (12.5ñ50) (12.5-25) (6.5ñ25)

C. paratropicalis 28.8 ± 14.8 21.3 ± 5.8 19.5 ± 5.7 NSstrains (n = 10) (12.5ñ50) (12.5-5) (12.5-50)

Candida species 20 ± 6.1 17.5 ± 6.1 15.1 ± 8.4strains (n = 5) (12.5ñ25) (12.5ñ25) (6.5ñ25)

Dermatophytes SA mediumstrains MIC (Mean value/ Range; µg/mL)

After 5 days After 15 days

Trichophyton 10.9 ± 2.04 (6.25ñ12.5) 21.9 ± 3.8 (12.5ñ25) NSmentagrophytes

varietas granulosum(n = 8)

Trichophyton 13.3 ± 5.5 (3ñ12.5) 20.3 ± 6.1 (12.5ñ25) NSmentagrophytes

varietas interdigitale(n = 8)

Epidermophyton 12.05 ± 0 (12.5ñ12.5) 25 ± 0 (25ñ25) NSfloccosum (n = 3)

SA mediumMolds strains MIC (Mean value/ Range; µg/mL)

After 5 days

Penicillium species 58.3 ± 70.1 (25ñ100) NA(n = 6)

Aspergillus species 50 ± 75.7(25ñ100) NA(n = 5)

Aspergillus nidulans 97.8 ± 73.2 (50ñ100) NA(n = 4)

Cladosporium species 50 ± 0 (50ñ50) NA(n = 3)

Mucor species 50 ± 0 (50ñ50) NA(n = 3)

SA ñ Sabouraudís medium; yeast nitrogen base (YNB); Roswell Park Memorial Institute (RPMI) medium; NS ñ not significant; NA ñ notapplicable.


the DNTDB were inoculated with 20 µL of moldssuspension. Plates were incubated at 27OC and readafter 5 days.

The enzymatic activity of the yeast-like fungiwas performed by API ZYM test (BioMerieux,Warsaw, Poland). (Table 1). This method allows thesystematic and rapid study of 19 enzymatic reac-tions using only very small sample quantities. Allprocedures were done according to the manufactur-erís instructions.

Strains were biotyped according to William-sonís classification (16) distinguishing 8 biotypes(A to H) based on the analysis of five enzymes:esterase (II), valine arylamidase (VI), naphtholphosphohydrolase (XI), a-glucosidase (XV), and N-acetyl-β-D-glucosidase (XVII). Additional biotypes(I to N) described by Kurnatowska and Kurnatowski(17) as well as biotypes described by Krajewska-Ku≥ak et al. (5), by Batura-Gabryel (18), and Bajeret al. (19) were also included in the assessment(Table 2).

We evaluated the effects of DNTDB on theultrastructure of the cells of C. albicans using 900OPTON transmission electron microscope at a mag-

nification of 12 000◊. For these strains the MIC ofDNTDB was 6.5 mg/mL.


Student t (two-tailed) test was used to comparemean MIC values, Wilcoxonís paired test was usedto compare enzymatic activity before and afterexposure of sample in sore scale.

Significance was defined as a p value of 0.05.These analyses were performed using statistics soft-ware (Statistica 10.0 PL).

RESULTS

MICs of the DNTDB values against the yeast-like fungi strains were ranging from 3 to 0 µg/mL,for dermatophytes from 3 to 25 µg/mL, and formolds from 25 to 100 µg/mL on the different media(Table 3). Mean MIC values for C. albicans onYNB and RPMI differed significantly (p < 0.001)when compared to SA (Table 3). Mean MIC valuesfor non C. albicans on YNB and RPMI differed sig-nificantly (p < 0.01) when compared to SA medium.Testing of 19 dermatophytes clinical isolates con-

Table 4. General biotype distribution before and after exposure to 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarboth-ioamide (DTNDB).

Reference C. albicans 10 231 C. albicans Non C. albicansBIOTYPES ATCC n = 1 n = 200 n = 100

ENZYMATIC before after before after before after

exposure exposure exposure exposure exposure exposure

according to Williamson et al. (22)

A 1 (100%) 191 (95.5%) 82 (41%) 31 (31%)

B 15 (15%)

C 7 (3.5%)

D 18 (9%)

E 11 (5.5%) 42 (42%) 31 (31%)

F 39 (19.5%)

H 20 (10%)

according to Kurnatowska and Kurnatowski (11)

F 9 (4.5%)

M 27 (27%) 22 (22%)

N

according to Krajewska-Ku≥ak et al. [9] and Batura-Gabryel et al. (2)

S 1 (100%) 23 (11.5%)

acccording to Brajer et al. (3)

T 17 (17%)

New biotype

new 15 (15%)


Tab

le 5

. Enz

ymat

ic a

ctiv

ity o

f C

andi

dast

rain

s be

fore

and

aft

er e

xpos

ure

to 2

,4-d

ihyd

roxy

-N-(

3-th

ioxo

-3H

-1,2

,4-d

ithia

zol-

5-yl

)ben

zene

carb

othi

oam

ide

(DN

TD

B).

The

num

ber

Num

ber

of e

nzym

e / m

ean

valu

es o

f th

e en

zym

atic

act

ivity

(po

int s

cale

)of

act

ive

III

III

IVV

VI

VII

VII

IIX

XX

IX

IIX

III

XIV

XV

XV

IX

VII

XV

III

XIX

enzy

mes

C. a

lbic

ans

stra

ins

befo

re e

xpos

ure

n =

200

mea

n1.

22.

52.

60.

83.

71.

91.

90.

40.

32

1.8

00.

20

2.3

0.7

3.5

0.5

016

SD0.

40.

60.

60.

70.

90.

70.

80.

50.

40.

90.

70

0.9

00.

80.

91.

50.

50

C. a

lbic

ans

stra

ins

post

exp

osur

e n

= 20

0

mea

n0.

41.

81.

60.

23.

11.

20.

80.

60

0.7

0.9

00.

30

0.9

00.

90

013

SD0.

50.

60.

60.

40.

970.

40.

40.

20

0.6

0.7

01.

10

0.8

00.

990

0

refe

renc

e C

. alb

ican

s st

rain

s n

= 1

befo

re e

xpos

ure

13

31

34

22

01

31

00

22

30

014

afte

r e

xpos

ure

11

10

12

11

00

01

00

00

10

09

non-

C. a

lbic

ans

stra

ins

befo

re e

xpos

ure

n =

100

mea

n1.

81.

92.

2.0.

34.

10.

70.

50

01.

62.

10

1.2

0.6

0.9

00.

30

013

SD1.

2.2

0.8

0.5

0.8

0.4

0.5

00

0.9

0.8

01.

80.

91.

40

0.5

00

non-

C. a

lbic

ans

stra

ins

post

exp

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= 10

0

mea

n1.

10.

81.

10

2.1

0.5

00

00.

61.

40

0.2

0.2

0.2

00.

20

011

SD0.

80.

70.

60

1.1

0.5

00

00.

50.

50

0.4

0.4

0.4

00.

40

0


Figure 2. Effects of 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide DNTDB on the ultrastructure of the cellsof C. albicans using transmission electron microscope at a magnification of 12 000◊. a, b - C. albicans cells before exposure to DNTDB;c, d - C. albicans cells after exposure to DNTDB

firmed some antifungal activity of the DNTDB.Testing of 21 molds clinical isolates confirmedsome antifungal activity of the DNTDB on SAmedium after 5 days of incubation against somemolds.

The reference C. albicans strains had the enzy-matic activity of 14 enzymes. Exposure to DNTDBinhibited the enzymatic activity of nine enzymes(Table 4). The 200 C. albicans strains had the enzy-matic activity with 16 enzymes. Exposure to DNTDinhibited the enzymatic activity of 13 enzymes. Thehighest enzymatic activity before and after exposurecontained leucine arylamidase and N-acetyl-β-glu-cosaminidase. The 100 non C. albicans strains had theenzymatic activity of 13 enzymes. Exposure to DNTDinhibited the enzymatic activity of 11 enzymes.

Two hundred pre-exposure C. albicans strainsdisplayed 95.5% activity for biotype A and 4.5% forbiotype F; after exposure seven different biotypeswere observed with biotypes A (41%) and F (19.5%)present in the highest quantities (Table 3). One hun-dred non C. albicans pre-exposure strains exhibitedactivity for biotypes A (31%), E (42%) and M(27%), and after exposure, four different biotypeswere seen, with E (31%) being the predominant bio-type. (details are shown in Table 4).

After exposing 15 non C. albicans strains toDNTDB, it was not possible to classify the remainsto known biotypes. (Table 5).

In the case of C. albicans 10 231 ATCC strain,biotype A changed to biotype S after exposure toDNTDB. Also, 41% of C. albicans strains changedto biotype A. Only 14% of C. albicans did notchange to biotype E. (details are shown in Table 6.) In the control group, C. albicans cells were round oroval with a smooth, thick wall and were of differentsizes (small, medium, or large) (Fig. 2). In theDNTDB group, C. albicans cells had a corrugatedsurface because of its thinner wall with a changed,irregular shape. A few budding cells were observedto have an abnormal sprouting interrupting the con-tinuity of the cell walls and thinning the cellsí cyto-plasm. This is known as selected cell destructionand autolysis.

DISCUSSION

The study demonstrated a potential antifungalactivity of new 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide againstC. albicans, dermatophytes and molds in vitro. TheMICs values were lower compared with currentlyused antifungal drugs (itraconazole and flucona-zole). Furthermore, C. albicans strains used in thisstudy were isolated from patients and were resistantfor several antimycotics.

Among factors known to contribute to thepathogenicity of yeast, enzymes play a significant


role, possibly being harmful to host tissues whenthey are liberated by the fungi. A correlation hasbeen demonstrated between the amount of phospho-lipase produced and virulence in C. albicans strainsand other yeast species (7). In the present study, theDNTDB inhibited the enzymatic activity of Candidastrains. Our results are in accordance with previousstudies (20-22).

Bujdakova et al. (20) assessed anti-Candidaactivity of 6-amino-2-n-pentylthiobenzothiazole,benzylester of (6-amino-2-benzothiazolylthio)aceticacid and of 3-butylthio-(1,2,4-triazolo)-2,3-benzoth-iazole and compared to that of 2-mercaptobenzoth-iazole. They found that these compounds wereactive against Candida strains.

Kucukbay and Durmaz (22) assessed fortyorganic or organometallic derivatives of benzimida-zole and benzothiazole for their in vitro antifungalactivity against C. albicans. Four of the tested com-pounds were found effective at the MICs between400-600 µg/mL.

Cetinkaya et al., (21) assessed azolium saltsand neutral 2-aryl derivatives of benzimidazole,benzothiazole and benzoxazole in vitro antimicro-bial activity against standard strains: E. faecalis, S.aureus, E. coli, P. aeruginosa, C. albicans, and C.tropicals. The tested compounds showed also anti-fungal activity against C. albicans and C. tropicals,with the MICs ranging between 50-200 µg/mL.

From these data, we can conclude that DNTDBis capable of inhibiting both the yeasts, molds, anddermatophytes in vitro. Furthermore, we used C.albicans strains for tests which were resistant forseveral antimycotics. The results also suggest thatthe action of DNTDB affects the structure of thefungal cell wall.

Conflict of interest

All authors report no conflicts of interest with-in this manuscript.

Table 6. Change biotypes of Candida strains after exposure to 2,4-dihydroxy-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)benzenecarbothioamide(DNTDB).

Biotype change Reference C. albicans C. albicans Non C. albicans enzymatic 10 231 ATCC n = 1 n = 200 n = 100

before exposure after exposure

to DNTDB to DNTDB N % N % N

%

A A 82 41%

A B

A C 7 3.5% 11 11%

A D 18 9%

A E 11 5.5% 12 12%

A F 39 19,5%

A H 20 10%

F F 9 4.5%

A S 1 100% 23 11.5%

A T 5 5%

E B 3 3%

E E 14 14%

E M 12 12%

E T 11 11%

M B 1 1%

M E 5 5%

M M 10 10%

M T 1 1%

A new 3 3%

E new 2 2%

M new 10 10%


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Received: 12. 11. 2015


As life expectancies increase worldwide andpopulation ages, avascular necrosis of the femoralhead (ANFH) has become a much more commoncondition (1, 2), and is listed as the third largest geri-atric disease by the World Health Organization(WHO). As a result of its high rate of morbidity anddisability, ANFH imposes significant financial bur-

dens to patients, families, and healthcare systems.Current treatment of ANFH includes surgery andmedication (3). Due to the serious trauma, longrecovery period, high medical costs, and the risk ofside effects of the surgery and medication associat-ed with conventional ANFH treatment (4), newtreatment options that effectively prevent, diminish,

EFFECT OF COLLA CORNUS CERVI COMBINED WITH LV-MEDIATEDBMP7 TRANSFECTED BMSCs ON ANFH IN RATS

PING WANG1,2, BIN SHI3, ZHI-HUI GAO4, TIE-FENG SUN4, WU-BIN YANG5, SHU-FANG HAN6,PENG LIU6, LEI-LEI WANG7, BO-NIAN ZHAO2, and DAN-DAN WANG3*

1School of Precision Instrument and Opto Electronics Engineering, Tianjin University, Tianjin 300072, Peopleís Republic of China

2Shandong Academy of Chinese Medicine, Jinan 250014, Peopleís Republic of China3Shandong Medicinal Biotechnology Centre, Shandong Academy of Medical Sciences,

Jinan 250062, Peopleís Republic of China4Shandong University of Traditional Chinese Medicine,

Jinan 250355, Peopleís Republic of China5Chongqing Cancer Hospital, Chongqing 400030, Peopleís Republic of China

6The General Hospital of Jinan Military Command, Jinan 250012, Peopleís Republic of China

7Ecology Institute of Shandong Academy of Sciences, Jinan 250014, Peopleís Republic of China

Abstract: In the present study, we investigated the combined effect of Colla Cornus Cervi (CCC) and BMP7-overexpressing bone marrow-derived mesenchymal stem cells (BMSCs) on osteogenic induction and the treat-ment of avascular necrosis of the femoral head (ANFH). BMSCs were isolated from rats. BMP7-overexpress-ing BMSCs were generated by lentiviral-mediated gene transduction. Cell proliferation, alkaline phosphatase(ALP) activity, osteogenesis related gene expression, osteocalcin levels, and calcified nodules were quantifiedand compared between four groups: untreated controls, BMSCs cultured with CCC complex medium, BMP7-overexpressing BMSCs, and BMP7-overexpressing BMSCs cultured with CCC complex medium(CCC+BMP7). CCC+BMP7 BMSCs showed higher proliferation rate. ALP activity and osteaocalcin contentwere significantly increased in CCC+BMP7 BMSCs. The osteogenesis related genes, COLI, and integrin-α2, -α5, and -fl1, were expressed significantly higher in CCC+BMP7 BMSCs. The number of calcified nodules inthe CCC+BMP7 group was significantly higher than that in other groups. For in vivo assays, ANFH wasinduced in rats, and BMSCs were injected into the femoral head of the lower left extremity. In rats with inducedANFH, general observation scores of the CCC+BMP7 injected group were significantly higher than the modelgroup. X-ray and microscopic observations revealed that ANFH was significantly improved and femoral headcells gradually recovered in rats treated with CCC+BMP7 BMSCs. Our results suggest that CCC+BMP7 sig-nificantly promote the proliferation and osteogenic differentiation of BMSCs in vitro. CCC+BMP7 BMSCs pro-mote the ability of repairing ANFH in rats, providing a new therapeutic paradigm for the treatment of ANFH.

Keywords: Colla Cornus Cervi, BMP7, bone marrow-derived mesenchymal stem cells, avascular necrosis ofthe femoral head, osteogenic differentiation, treatment

1521

* Corresponding author: e-mail: [email protected]; phone: +86-531-82629331; fax: +86-531-82951586

1522 PING WANG et al.

or delay the development of ANFH, and that obviatethe need for invasive surgery, are of pivotal impor-tance.

ANFH is a pathological process caused byinterruption of blood supply to the bone, resulting inthe eventual collapse of bone structure if not reme-died in time (5, 6). Recently, enhancement of bonerepair and regeneration in the necrotic zone by bone-marrow-derived mesenchymal stem cells (BMSCs)has been highlighted for the treatment ofosteonecrosis prior to collapse of the head (7, 8).

Deer antler (Cervus elaphus Linn., antler) is ahighly valued traditional Chinese medicine, and haslong been considered to be beneficial in the preven-tion and treatment of various diseases, such as acuteand chronic arthritis, osteoporosis, and fractures, asevidenced in animal models and human clinical trials(9). Each spring, the hard antler from the previousseason is cast off and the antler grows anew atincredible speeds (10); this growth includes vascular,connective tissues, and cartilage, which suggests thatthe active compounds in antler can promote and sus-tain bone formation and growth (11). Colla CornusCervi is a refined extract from deer antlers obtainedby boiling the antler in water and concentrating theextract. In the present study, BMSCs were isolatedand cultured, and BMSCs overexpressing humanbone morphogenetic protein 7 (BMP7) were generat-ed using lentiviral transduction methods. We investi-gated the combined effect of CCC and overexpres-sion of BMP7 in BMSCs on osteogenic inductionand the treatment of ANFH in vitro and in vivo.

EXPERIMENTAL

Animals and reagents

Wistar rats weighting 180~220 g (License No.scxk [Lu] 20130009) and 8 days old Sprague-Dawley rats weighting 25~35 g (License No. scxk(Lu) 20130009) were purchased from ShandongUniversity Laboratory Animal Center. All animalexperiments were approved by the InstitutionalAnimal Care and Use Committee of ShandongAcademy of Chinese Medicine. The experimentswere conducted according to the National ResearchCouncil's guidelines.

Deer antler tablets were purchased fromShandong Baiweitang Chinese Herbal MedicineDrinks Slice Company Limited. Quality manage-ment of deer antler tablets was performed byDepartment of Pharmacognosy of ShandongAcademy of Chinese Medicine. Quality control wasconducted in accordance with the regulations of theChinese Pharmacopoeia 2010 Edition.

The reagents used in experiments were as fol-lows: Dulbecco's modified Eagle's medium(DMEM-LG; Hyclone, USA), fetal bovine serum(FBS; Hyclone, USA), polybrene (Sigma-Aldrich,St. Louis, MO), phosphate buffered saline solution(PBS; Solarbio, Beijing, China), TRIzol (Solarbio,Beijing, China), fluorescence labeled mouse anti-ratmonoclonal antibodies (CD34-FITC, CD45-FITC,and CD90-FITC, SinaSun, Beijing, China), primaryantibodies used in western blotting (Santa Cruz,USA), secondary antibodies used in western blotting(Beijing Chinese Fir Golden Bridge BiotechnologyCo. Ltd., China), Cell Counting Kit-8 (CCK-8,Boster, Wuhan, China), colorimetric assay kit(Nanjing Jian Cheng Bioengineering Institute,Nanjing, China), enzyme-linked immunosorbentassay (ELISA) kit (Nanjing Jian ChengBioengineering Institute), ReverTra Ace qPCR RTKit (TOYOBA, Japan), and SYBR Green mastermix (Invitrogen, Carlsbad, California, USA).

CCC complex medium preparation

Deer antler tablets were pulverized and mixedwith 4-5 times (w/w) distilled water in a round-bot-tom flask. The antler tablets were boiled and juiceswere taken every 3 h. Supplemented distilled waterand boiled until antler tablets were soft and could bepinched into powder. The juices were gathered andconcentrated in vacuo at 50OC until the water con-tent was 11% (w/w%). The extracts were filteredwith double gauze. CCC was obtained after coolingto room temperature.

CCC quality identification was conducted bythin-layer chromatography (TLC) according to theregulations of the Chinese Pharmacopoeia 2010Edition. Briefly, 1 g CCC was added into 10 mL of70% ethanol, and treated with ultrasonics for 15 min.After filtration, filtrates were obtained as the experi-mental sample. The control sample was 0.5 mg/mLglycine standard substance (glycine dissolved in 70%ethanol). The two samples were spotted on a same sil-ica gel TLC plate, respectively, and then dipped in thedeveloping solvent (N-butyl alcohol, glacial aceticacid and water, 3 : 1 : 1, v/v/v). The developing sol-vent traveled up the plate until 1 cm from the top.Then, the plate was taken out and dried in the air. Thespots on the plate were detected by ninhydrin solution.

The CCC were sterilized by γ-ray radiation for2 h (accumulated 105 rad), and then prepared forCCC complex medium. The CCC complex mediumwas low glucose Dulbecco's modified Eagle's medi-um (DMEM-LG) containing 0.25% CCC (w/v%),20% FBS, 100 U/mL penicillin and 100 µg/mLstreptomycin.

Effect of Colla Cornus Cervi combined with LV-mediated BMP7 transfected... 1523

Recombinant lentivirus construction and infec-

tion

Lentiviral vectors (LV) for overexpression ofrecombinant BMP7 (PLV-sfGFP(2A)-BMP7) andempty control vector (PLV-sfGFP(2A)-puro) wereobtained from Inovogen Tech Company (Beijing,China).

LV transfection was performed by adding virussolution to BMSCs in the presence of 4 µg/mL poly-brene. Forty-eight hours after infection, the cellswere selected in complete culture medium contain-ing 300 µg/mL puromycin. Puromycin-resistantcells were pooled and cultured for western blotdetection of recombinant BMP7. Stable cell lineswere cultured for further analysis.

BMSCs culture and characterization

Eight-days-old rats were sacrificed, and bonemarrow was flushed out from femur and tibia withDMEM-LG under aseptic conditions. After diges-tion, cells were harvested through a cell strainer(100 micron mesh) and centrifuged (1200 rpm, 5min). Cells were placed in a 25-cm2 flask and cul-tured in DMEM supplemented with 20% FBS at37OC in 5% CO2 in a cell culture incubator (GasaxyS, RS Biotech Co., UK).

The fourth generation of BMSCs were collect-ed and washed three times with PBS. After digestionwith 0.25% trypsin, single cell suspension was har-vested, and then, respectively, incubated with mono-clonal antibodies CD34-FITC, CD45-FITC, andCD90-FITC at room temperature for 30 min in thedark. After washing with PBS, cells were fixed with20 g/L paraformaldehyde and analyzed using a flowcytometer (Beckman, USA).

The BMSCs were randomly divided into fourgroups: the normal control group, CCC group(BMSCs cultured with CCC complex medium),BMP7 group (PLV-sfGFP(2A)-BMP7 transfectedcells), and CCC+BMP7 group (PLV-sfGFP(2A)-BMP7 transfected BMSCs cultured with CCC com-plex medium).

Western blot assays

Cells were collected and lysed in RIPA lysisbuffer, and the lysates were harvested by centrifuga-tion (12,000 rpm) at 4OC for 30 min. Then, 30 µgprotein per sample was loaded into a 12% sodiumdodecyl sulfate polyacrylamide gel for separation byelectrophoresis, and were then transferred onto apolyvinylidene fluoride membrane. Membraneswere blocked for 60 min with 5% non-fat milk, andincubated overnight at 4OC wiith the following pri-mary antibodies: rabbit anti-rat monoclonal anti-

body against ACTIN, rabbit anti-rat monoclonalantibody against VEGF, and goat anti-rat polyclon-al antibody against BMP7 (at a 1 : 200 dilution).After washing with TBST (Tris-buffered saline with1â Tween 20) for 10 min, the membranes wereprobed with the following horseradish peroxidase(HRP)-conjugated secondary antibodies: goat anti-rabbit IgG antibody and mouse anti-goat IgG anti-body at a 1 : 5000 dilution at 37OC for 1 h. Afterthree washes with TBST, the membranes weredeveloped by an enhanced chemiluminescence sys-tem (Cell Signaling Technology, Danvers,Massachusetts, USA).

Proliferation assay

The CCK-8 kit was used to evaluate the growthrate and relative activity of cells according to themanufacturer's protocol. The BMSCs were random-ly divided into four groups: the normal controlgroup, CCC group, BMP7 group, and CCC+BMP7group. Briefly, cells were seeded in a 96-well plateat a density of 5 ◊ 103 cells per well. After incuba-tion for 24 h, 48 h, and 72 h, 10 µL CCK-8 wasadded to each well and incubated for additional 4 h.The absorbance at 450 nm was measured with amicroplate reader. Each experiment was performed intriplicate. The cell activity was calculated as follows:cell activity = (OD experimental group - OD blank control group) /(OD control group - OD blank control group) ◊100.

Analysis of alkaline phosphatase (ALP) activity

ALP activity was measured using a colorimet-ric assay kit following the manufacturer's instruc-tions. The BMSCs were randomly divided into fourgroups: the normal control group, CCC group,BMP7 group, and CCC+BMP7 group. Briefly, afterincubation for 48 h, the medium in the 12-well platewas removed and the cells were washed three timeswith PBS. Cell lysis was accomplished by adding 20µL 0.05% Triton X-100 to each well and incubatingfor 5 min at room temperature. The absorbance at520 nm, indicative of ALP activity, was measuredwith a microplate reader. The total protein concen-tration was measured with a BCA kit (Nanjing JianCheng Bioengineering Institute) according to themanufacturer's protocol. The ALP activity was cal-culated as follows: ALP activity (King unit/gramprotein (gprot)) = (ODsample - ODblank control)/ (ODstandard

- ODblank control) ◊ phenol standard concentration (0.02mg/mL) - total protein concentration (gprot/mL).

Determination of osteocalcin levels

Osteocalcin determination was performed byan ELISA kit following the manufacturer's instruc-


tions. BMSC media were collected every 3 days andfrozen at -20OC for further analysis. The standardcurve was protracted according to the absorbance ofstandard samples measured at 450 nm with amicroplate reader. Osteocalcin content was calculat-ed according to the standard curve.

Staining of calcified nodules

CCC+BMP7 BMSCs were seeded in a 12-wellplate at a density of 5 ◊ 103 cells per well. Afterincubation for 21 days, the medium in the wells wasremoved and the cells were washed twice with PBS.The cells were fixed with 95% ethanol at 4OC for 10min. After three washes with double distilled water,

0.1% alizarin red-Tris HCl (pH 8.3) was added forstaining, and samples were incubated in a 37OCwater bath for 60 min. Stained cells were thenobserved under an inverted phase contrast micro-scope and calcified nodules were counted under lowpower magnification. Quantification of nodules wasperformed on five random fields per well.

Real time quantitative PCR (RT-qPCR)

Total RNA was extracted from BMSCs usingTRIzol according to the manufacturer's instructions.RNA concentration and quantity were assessed bymeasuring absorbance at 260 nm using a Nanodropspectrophotometer (ND-1000, Thermo Scientific,

Table 2. General observation table.

Observation item Index Specific performance Score

Not 7

Behavior function Limp Mild 5

Moderate 3

Serious 0

> 70O 9

Flexion > 60O 5

> 30O 2

< 30O 0

> 30O 4

Joint range Abduction > 15O 2

of motion > 5O 1

< 5O 0

> 15O 2

Pronation > 5O 1

< 5O 0

> 15O 2

Supination > 5O 1

< 5O 0

Table 1. Primer sequences and PCR products size.

Gene Primer PCR products size

COL Iforward 5'-GGACTTGGGCAAGACAGTGATC-3'reverse 5'-GTCACGTTCAGTTGGTCAAAGAT-3'

333 bp

Integrin α2forward 5'-CAGTGAGAGCCAAGAAACAA-3'reverse 5'-AACCATAGCCAACAGCAAA -3'

665 bp

Integrin α5forward 5'-CTTCGGTTCACTGTTCCTC -3'reverse 5'-TGGCTTCAGGGCATTT-3'

508 bp

Integrin fl1forward 5'-ACAGAAGAAGTAGAGGTGGTC-3'reverse 5'- GAGGTTGAAATGGGAGC-3'

660 bp

fl-Actinforward 5'-GCTTACATGTCTCGATCCCACTTAA-3'reverse 5'-CTCGCGCTACTCTCTCTTTCTGG-3'

333 bp


USA). First-strand cDNA synthesis was performedusing a ReverTra Ace qPCR RT Kit according to themanufacturer's recommendations. RT-qPCR wasemployed to evaluate the relative mRNA expressionof COLI, integrin α2, integrin α5, integrin fl1, andfl-actin (as an internal control). The primers andPCR products size are shown in Table 1. Gene-spe-cific amplification was performed using an ABI7900HT real-time PCR system (Bio-Rad,American) with a 15-µL PCR mix containing 0.5 µLof cDNA, 7.5 µL of 2 ◊ SYBR Green master mix,and 200 nM of the appropriate oligonucleotideprimers. The cycling parameters are as follows:95OC for 15 min, followed by 45 cycles of 95OC for30 s and 60OC for 1 min. The resolution curve wasmeasured at 95OC for 15 s, 60OC for 15 s and 95OCfor 15 s. Ct values were calculated from the thresh-old cycles with the instrument's software (SDS 2.3),and the relative expression of mRNA was normal-ized against the results for fl-actin. Data were ana-lyzed using the comparative threshold cycle (2-∆∆CT)method.

Transplantation of BMSCs

After 1 week of acclimatization, the 30 Wistarrats weighting 180~220 g were randomly divided

into two groups: the control group and the ANFHmodel group. The rats of ANFH model group weregiven 70 mg/kg retinoic acid by oral gavage onceper day for 6 weeks. After 6 weeks, two rats wererandomly selected from the ANFH group to confirmwhether the treatment with retinoic acid successful-ly induced ANFH. PLV-sfGFP(2A)-BMP7 trans-fected BMSCs cultured in CCC complex mediumwere resuspended at 6 ◊ 106 cells/mL in normalsaline, and injected into the necrotic femoral head ofthe left lower extremity. As a model control, thenecrotic femoral head of right lower extremity didnot receive any treatment in the ANFH group. After4, 12, and 24 weeks, general observation, femoralhead gross observation, X-ray observation, andpathological examination of the femoral head wereperformed. The general observation and score wereconducted according to Table 2.


All experiments were performed in triplicate.All statistical analyses were performed using theStatistical Package for the Social Sciences, version16.0 (SPSS Inc., Chicago, IL, USA). The compari-son of multiple means was analyzed using the F test.The comparison of the rate was analyzed using the

Figure 1. A: Results of Giemsa staining of BMSCs (200 ◊ magnification). B: Western blotting results of BMP7 expression in LV-mediat-ed BMSCs. C: Refined CCC products. D: Quality identification of CCC products by TLC. 1: glycine standard. 2: CCC sample


σ2 test. The results were presented as the mean ±standard deviation (SD). A two-sided p-value lessthan 0.05 was considered to be statistically signifi-cant.

RESULTS

BMSCs identification

Prior to their use in other experiments, culturedBMSCs were characterized. Results of Giemsastaining showed triangular or fusiform cells with fullcytoplasm and clearly visible nucleoli stained uni-formly (Fig. 1A). The expression of CD34, CD45and CD90 was detected in BMSC populations byflow cytometry. Results showed that more than 90%of BMSCs were CD90+, while fewer than 5% ofBMCs were CD34+ or CD45+. The percentages ofpositively stained BMSCs were as follows: CD34+

cells, 2.65 ± 0.56%; CD29+ cells, 96.87 ± 2.54%;CD90+ cells, 90.37 ± 3.55%; and CD45+ cells, 1.62± 0.16%.

BMP7 expression analyzed by western blotting

BMSCs were transfected with recombinantPLV-sfGFP(2A)-BMP7 and empty control vectorPLV-sfGFP(2A)-puro Following selection of stabletransductants BMP7 expression was detected usingwestern blotting. The results showed a BMP7 bandat the expected size and the amount of BMP7 pro-tein present was further measured by densitometry(statistical analysis of significance showed p <0.05). BMP7 protein expression was markedlyincreased in the PLV-sfGFP(2A)-BMP7 transfected

cells compared with control vector PLV-sfGFP(2A)-puro transfected cells (Fig. 1B).

Characteristics of Colla Cornus Cervi

The CCC product was yellowish-brown,translucence, crisp and fragile, with a smooth sec-tion profile (Fig. 1C). The moisture content of CCCwas 11%, which was determined according toChinese Pharmacopoeia 2010 Edition (Appendix IXH, the first method). Quality identification showedthat the CCC displayed the same spots as glycinestandard at the corresponding position of TLC plate(Fig. 1D).

The role of CCC and BMP7 in BMSC prolifera-

tion

The CCK-8 proliferation assay revealed thatproliferation rate of the CCC, BMP7, andCCC+BMP7 BMSC groups was significantlygreater than the control BMSCs (Fig. 2A).Additionally, the proliferation rate of CCC+BMP7group was significantly higher than either the CCCor BMP7 groups. These results suggested that CCCcombined with BMP7 can enhance BMSC prolifer-ation.

Alkaline phosphatase activity

The activity of ALP was significantlyincreased in CCC+BMP7 BMSCs compared withthe CCC, BMP7, and control BMSC groups (p <0.05, Fig. 2B). There was no significant differencein ALP activity between CCC group and BMP7group. These results indicated that CCC canenhance the production of ALP in BMP7-overex-pressing BMSCs.

Osteocalcin determination

After osteogenic induction of BMSCs, theosteocalcin content increased over time. The osteo-calcin content in the CCC, BMP7, and CCC+BMP7BMSC groups was significantly higher than the con-trol BMSC group (Fig. 2C). The osteocalcin contentin CCC+BMP7 group was significantly higher than

Table 3. The mRNA expression of osteogenesis related genes (xñ ± s).

Group COL I Integrin α2 Integrin α5 Integrin β1

Control 5.9 ◊ 10-3 ± 9.9 ◊ 10-4 6.1 ◊ 10-8 ± 3.5 ◊ 10-8 6.4 ◊ 10-7 ± 1.2 ◊ 10-7 1.2 ◊ 10-7 ± 0.6 ◊ 10-7

CCC 1.5 ◊ 10-1 ± 2.5 ◊ 10-2# 1.1 ◊ 10-5 ± 0.5 ◊ 10-6# 3.2 ◊ 10-5 ± 7.5 ◊ 10-6# 2.5 ◊ 10-4 ± 0.9 ◊ 10-5#

BMP7 1.9 ◊ 10-1 ± 1.7 ◊ 10-2# 1.3 ◊ 10-5 ± 6.5 ◊ 10-6# 1.4 ◊ 10-5 ± 3.8 ◊ 10-6# 3.6 ◊ 10-4 ± 0.6 ◊ 10-5#

CCC+BMP7 2.6 ◊ 10-1 ± 5.6 ◊ 10-2* 1.5 ◊ 10-4 ± 4.0 ◊ 10-5* 2.4 ◊ 10-3 ± 0.6 ◊ 10-4* 2.2◊10-3 ± 1.1 ◊ 10-3*

* p < 0.01, # p < 0.05, compared with control group.; p < 0.05, compared with CCC or BMP7 group.

Table 4. Comprehensive score of general observation.

Group n Score

Blank control 10 22.20 ±1.32

Model control 10 4.60 ±1.35

CCC+BMP7 10 13.60 ±1.90 ##

n: numbers of cases in each group. ## p < 0.01, compared withmodel group.


that in other groups (p < 0.05), which suggested thatCCC can enhance osteoblastic differentiation ofBMP7-overexpressing BMSCs.

Calcified nodules staining

Results of alizarin red staining showed thatthere were no calcified nodules in blank controlgroup (Fig. 2D). The number of calcified nodules inCCC group and BMP7 group was significantly lessthan that in CCC+BMP7 group (Fig. 2D); the stain-ing intensity in CCC+BMP7 group was alsostronger than that in the other groups.

mRNA expression of osteoblast collagen type I

(COLI), integrin αα2, integrin αα5 and integrin fl1

At the seventh day after infection with lentiviralvectors, the mRNA expression of the osteogenesisrelated genes COLI, integrin α2, α5 and fl1 weredetermined by RT-qPCR. Expression of COLI, inte-grin α2, α5 and fl1 were significantly higher inCCC+BMP7 group compared with other groups(Table 3, p < 0.05). The expression of these genes inCCC group and BMP7 group were significantly high-er than in the control BMSC group (Table 3, p < 0.05).

The function of CCC and BMP7 on repair of

ANFH

Results of general observation were recordedand scored in Table 4. Results showed that score ofCCC+BMP7 group was significantly higher than themodel group (Table 4, p < 0.01).

X-ray observation

The necrotic femoral head of the left lowerextremity in ANFH model group showed cysticdegeneration and osteosclerosis, and some animalsdisplayed segmental femoral head flattening or sub-chondral collapse (Fig. 3Ab). X-ray film showedsmooth articular surface, and restoring necrotic andcystic area (Fig. 3Aa). Significant alleviation offemoral head necrosis was observed in the rats treat-ed with CCC+BMP7 BMSCs (Fig. 3A).

Microscopic observation

Femoral head samples obtained from controlrats showed normal ultrastructure (Fig. 3B). In themodel group, femoral heads exhibited osteocytedegeneration and necrosis, cell surface collapse, mar-row cavity bleeding, and synovial necrosis (Fig. 3B).

Figure 2. The effect of CCC and BMP7 on BMSCs. A: Proliferation of BMSCs. * p < 0.05, # p < 0.01, compared with normal controlgroup. °˜ p < 0.05, compared with CCC or BMP7 group. B: ALP activity. * p < 0.05, ** p < 0.01, compared with normal control group.# p < 0.05, compared with CCC or BMP7 group. C: Osteocalcin content. * p < 0.01, # p < 0.05, compared with normal control group. °˜

p < 0.05, compared with CCC or BMP7 group. D: Results of calcified nodules staining in BMSCs (200◊ magnification)


In the CCC+BMP7 group, the femoral head cellswere gradually recovered. Samples showed smoothfibrous cartilage and articular cartilage, and little car-tilage cell destruction (Fig. 3B). Our results suggestthat CCC and BMP7 play important functions in theability of BMSCs to repair ANFH in rats.


As world's population aging, ANFH hasbecome a widespread disease worldwide. Due tohigh rate of morbidity and disability, ANFH bringhuge financial burdens to patients and family.Presently, less-invasive methods replacing arthro-plasty are becoming a new trend in the treatment ofANFH. Stem cell therapies provide a new avenuefor the treatment of ANFH (7, 12). Deer antler ishighly valued in traditional Chinese medicine, andhas been used for over 2000 years (13). CCC, therefined extract from deer antler, is thought to havebone-invigorating therapeutic effects. BMP7, amember of bone morphogenetic proteins family, hasa putative role in early development and bone induc-tive activity (14). In the present study, we investi-

gated the effect of CCC combined with BMP7-over-expressing BMSCs on osteogenic induction andtreatment of ANFH both in vitro and in vivo.

CCC was decocted from cervidae animal sikadeer (Cevus Nippon Tem - minck) or red deer (C.elaphus L.). CCC glue is yellowish brown or redbrown, translucent, some upside has yellow whitefoam layer. CCC is thought to have the function ofwarming and tonifying the liver and kidney, replen-ishing vital essence and blood, and has been com-monly used in the prevention and treatment of osteo-porosis, to increase bone mineral content and densi-ty (10, 15). Modern chemical research indicates thatCCC contains 82.49% protein and more trace ele-ments, which are the material bases of replenishingvital essence and blood (9). Recently, CCC is usedin the treatment of osteoporosis and osteonecrosis ofthe femoral head. In support of this, our study foundthat the proliferation and osteogenic differentiationof BMSCs treated with CCC were significantlyenhanced compared to control BMSCs.

BMP7, a member of the transforming growthfactor fl family, has many biological functions,including regulating cell growth, proliferation, dif-

Figure 3. The combined effect of CCC and BMP7 BMSCs on the treatment of ANFH in rats. A: X-ray observation of rat femoral head. a - CCC+BMP7; b - ANFH model control. B: Pathological observation of the femoral head (200◊ magnification)


ferentiation, apoptosis, and in inducing bone forma-tion. A study by Jin Dan et al. revealed that overex-pression of BMP7 significantly promoted theosteogenic differentiation of BMSCs (16). KangYan et al. found that BMP7 enhances osteogenicdifferentiation of BMSCs transfected with a BMP7-recombinant-vector (17). Adeno-associated virusmediated VEGF and BMP7 gene transfer stimulatesangiogenesis and bone regeneration, which may bea new therapeutic technique for the treatment ofANFH (18). These studies prompted us to considerBMP-7 overexpressing BMSCs as ideal seed cellsfor engineering bone tissue remodeling. Consistentwith these findings, our research revealed that over-expression of BMP7 through lentiviral transductionof PLV-sfGFP(2A)-BMP7 vector promoted the pro-liferation and osteogenic differentiation of BMSCsin rats, indicating that BMP7 can induce undifferen-tiated mesenchymal cells to differentiate into chon-drocytes or osteoblasts.

In the current study, cell proliferation assaysshowed that the growth rate of CCC+BMP7 BMSCgroup was significantly higher than CCC or BMP7groups. Compared with the CCC, BMP7, controlgroups, the activity of ALP was significantlyincreased in CCC+BMP7 BMSCs. Additionally, thecontent of osteocalcin in CCC+BMP7 group wassignificantly higher than that in other groups. ThemRNA expression of osteogenesis related genesCOLI, integrin α2, α5 and fl1 was significantlyhigher in CCC+BMP7 group compared with othergroups. These results suggested that CCC can sig-nificantly promote the proliferation and differentia-tion of BMP7-overexpressing BMSCs in vitro.

Additionally, we used a rat model of ANFHmodel to investigate the function of CCC combinedwith BMP7 on the ability of BMSCs to induce repairof ANFH in rats. Results of general observationwere recorded and scored, and indicated that thescore of ANFH rats treated with CCC+BMP7BMSCs was significantly higher than the controlgroup. Moreover, the number of calcified nodules inCCC+BMP7 group was significantly higher thanthat in other groups. X-ray and microscopy revealedthat the femoral head necrosis was significantlyrelieved and the femoral head cells were graduallyrecovered in the CCC+BMP7 BMSCs treatedANFH rats compared with the model group.

In conclusion, we have demonstrated that CCCand BMP7 can significantly promote the prolifera-tion and osteogenic differentiation of BMSCs.Furthermore, our results demonstrate that CCC andBMP7 play important functions on the ability ofBMSCs to facilitate repair of ANFH in rats, and

indicate that this stem cell therapy can favorablyenhance the repair of avascular necrosis of thefemoral head. Our research suggests that CCC com-bined with BMP7 may provide a new therapeuticstrategy in the use of BMSC and stem cell therapyfor the treatment of ANFH.


All authors have read and approved submissionof the manuscript. The authors declare that theyhave no competing interests.

Acknowledgments

This work was supported by the NationalNatural Science Foundation of China for financialsupporting (81273776), Shandong Science andDevelopment Program (2014GSF118026), Shan-dong Science and Development Program(2010GSF10278), Shandong Major Projects ofIndependent Innovation (2012ZHZX1C0405), JinanYouth Science and Technology Special StarProgram (Jike character (2012) 01-42) and Scienceand Technology Plan of Shandong Academy ofMedical Sciences (2014-48). The funders had norole in study design, data collection and analysis,decision to publish, or preparation of the manu-script.

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1. Zhang C., Zou Y.L., Ma J., Dang X.Q., WangK.Z.: BMC Musculoskelet. Disord. 16, 132(2015).

2. Herndon J.H., Aufranc O.E.: Clin. Orthop.Relat. Res. 86, 43 (1972).

3. Hong Y.C, Zhong H.M, Lin T, Shi J.B.: Int. J.Clin. Exp. Med. 8, 5207 (2015).

4. Martin J.R., Houdek M.T., Sierra R.J.: Croat.Med. J. 54, 219 (2013).

5. Shi B., Li G., Wang P., Yin W., Sun G., Wu Q.,Yu G.: J. Ethnopharmacol. 127, 124 (2010).

6. Shah K.N., Racine J., Jones L.C., Aaron R.K.:Curr. Rev. Musculoskelet. Med. 8, 201 (2015).

7. Li Z.H., Liao W., Cui X.L., Zhao Q., Liu M.,Chen Y.H.: Int. J. Med. Sci. 8, 74 (2011).

8. Mao Q., Jin H., Liao F., Xiao L., Chen D., TongP.: Bone 57, 509 (2013).

9. Yang H.O., Kim S.H., Cho S.H., Kim M.G.,Seo J.Y., Park J.S.: Chem. Pharm. Bull. 52, 874(2004).

10. Li C., Zhao H., Liu Z., McMahon C.: Int. J.Biochem. Cell Biol. 56, 111 (2014).


11. Ivankina N.F., Isay S.V., Busarova N.G.,Mischenko T.Ya.: Comp. Biochem. Physiol. B106, 159 (1993).

12. Wen Q., Zhou L., Zhou C., Zhou M., Luo W.,Ma L.: J. Cell. Mol. Med. 16, 1260 (2012).

13. Li M., Wang C.Y., Li S.D., Zhang Y., Zhang L.:Chinese Journal of Drug Evaluation 31, 310(2014).

14. Zhang C., Ma J., Li M., Li X.H., Dang X.Q.:Transl. Res. 166, 269 (2015).

15. Zhao Q.C., Kiyohara H., Nagai T., Yamada H.:Carbohyd. Res. 230, 361 (1992).

16. Jin D., Zeng W.S., Pei G.X., Hu B.S., Wei K.H.et al.: Medical Journal of Chinese People'sLiberation Army 27, 477 (2002).

17. Kang Y., Liao W.M., Yuan Z.H., Sheng P.Y.,Zhang L.J. et al.: Acta Pharmacol. Sin. 28, 839(2007).

18. Zhang C., Wang K.Z., Qiang H., Tang Y.L., LiQ., Li M.: Acta Pharmacol Sin. 31, 821 (2010).

Received: 24. 11. 2015


Lipoic acid, also known as α-lipoic acid orthioctic acid (LA, LA-(SS), 1,2-dithiolane-3-pen-tanoic acid) and its reduced form dihydrolipoic acid(DHLA, DHLA-(SH)2, 6,8-dimercaptooctanoicacid) are present in all prokaryotic and eukaryoticcells. The chemical structures of LA and DHLA areshown in Figure 1. LA is synthesized in mammals inthe mitochondria of the liver and other tissues. It issuggested that octanoic acid is the precursor of theeight-carbon fatty acid chain of LA while cysteine isthe source of sulfur. Most of LA in the body is foundtightly bound to proteins by an amide linkage(lipoamide, lipoyl group) to a lysine residue (lipolly-sine). This lipoamide bond functions mainly as acofactor of multienzymatic mitochondrial complex-es catalyzing oxidative decarboxylation of α-ketoacids (1, 2). The lipoyl group can be oxidizedand reduced by lipoamide dehydrogenase.

Apart from endogenous synthesis, LA is alsoabsorbed from food. LA has been detected in the

form of lipollysine in various natural products. Themost important dietary sources of it include spinach,broccoli and meat offal (3). Lipollysine absorbedfrom the diet can be hydrolyzed in blood by lipoami-dase. Moreover, LA has now become a commoningredient of different formulas for athletes, anti-

INACTIVATION OF ALDEHYDE DEHYDROGENASE BY NITROGLYCERININ THE PRESENCE AND ABSENCE OF LIPOIC ACID AND DIHYDROLIPOIC

ACID. IMPLICATIONS FOR THE PROBLEM OF DIFFERENTIAL EFFECTS OF LIPOIC ACID IN VITRO AND IN VIVO

ANNA BILSKA-WILKOSZ1, MAGDALENA G”RNY1, MAGDALENA DUDEK2, LESZEK NOWI—SKI2, MAREK BEDNARSKI3, MA£GORZATA ICIEK1,

DANUTA KOWALCZYK-PACHEL1, MARIA SOKO£OWSKA-JEØEWICZ1, BARBARA FILIPEK2

and LIDIA W£ODEK1*

1Chair of Medical Biochemistry, Jagiellonian University, Medical College, 7 Kopernika St., PL 31-034 KrakÛw, Poland

2Department of Pharmacodynamics, 3Department of Pharmacological Screening, Jagiellonian University,Medical College, 9 Medyczna St., PL 30-688 KrakÛw, Poland

Abstract: Lipoic acid (LA-(SS), LA) and its reduced form - dihydrolipoic acid DHLA-(SH)2, DHLA) are syn-thesized mainly in the mammalian liver. In this study, we investigated in vitro the inactivation of yeast alde-hyde dehydrogenase (ALDH) by nitroglycerin (GTN) in the presence and absence of LA and DHLA. In vivostudies were performed to answer the question whether LA administered jointly with GTN for 8 days will affectthe ALDH activity in the rat liver. The results indicated that in vitro both LA and DHLA restored and protect-ed ALDH activity against GTN-induced inactivation, while treatment of rats with LA and GTN in combinationdid not provide any protection against GTN-induced ALDH inhibition. In summary, the obtained results seemto confirm earlier reports indicating the differential effects of LA in vitro and in vivo.

Keywords: lipoic acid, dihydrolipoic acid, nitroglycerin, aldehyde dehydrogenase

1531

* Corresponding author: e-mail: [email protected]; phone: +48 12 4227400; fax: +48 12 4223272

Figure 1. Chemical structures of lipoic acid (LA) and its reducedform dihydrolipoic acid (DHLA)

1532 ANNA BILSKA-WILKOSZ et al.

aging supplements and even pet food (4). In recentyears, pharmacological properties of exogenouslysupplemented LA have attracted attention of manyresearchers. It is suggested that therapeutic effectsof LA supplementation can be attributed to thepotent antioxidant properties of LA and DHLA.

The reductive potential of the LA/DHLA cou-ple has been estimated at -320 mV (1) or -290 mV(5). This means that DHLA is able to reduce notonly reactive oxygen and nitrogen species (ROS andRNS, respectively) but also the oxidized forms ofother antioxidants, both hydrophilous, such as glu-tathione disulfide (GSSG) and dehydroascorbic acid(DHA), and hydrophobic, such as chromanoxyl rad-ical of tocopherol (TOH) (6ñ10) (Table 1). For this

reason, DHLA is often called the antioxidant ofantioxidants or a universal antioxidant (11). Theoxidized form, i.e., LA has also been demonstratedto express antioxidant properties (11, 12) (Table 2).

Redox reactions are fundamental processes inmetabolism of all cells. A disruption of the pro- andantioxidant balance can explain the mechanism ofetiopathogenesis and progression of many, appar-ently distant pathological states. This is the reasonfor an increasing significance of antioxidants inmedicine and more common use of the term ìoxida-tive stressî in medical sciences. However, since theactivity of LA and DHLA as direct scavengers ofROS/RNS was confirmed mostly by in vitro experi-mental studies, some authors question the efficacy

Table 2. The scavenging capacity of LA and DHLA for the selected ROS and RNS.

ROS/RNS LA DHLA Scavenging capacity Reference

O2ï- no yes - (11)

no no - (12)

H2O2

yes yes LA < DHLA (11)no yes - (12)

ïOH yes yes LA < DHLA (11)LA = DHLA (12)

ONOO- yes yes LA < DHLA (11, 12)

NOï yes yes LA < DHLA (11, 12)

Table 1. The values of standard biological redox potentials (E0` ) of selected redox systems

System E0' (V) Reference

Acetic acid/acetaldehyde -0.60 (11)

Acetylo-CoA/acetaldehyde -0.41 (11)

NAD/NADH + H+ -0.32 (12, 14)

LA/DHLA -0.29 (5, 12, 13)

GSSG/GSH -0.24 (12)

Cystine/cysteine -0.22 (12, 15)

Acetaldehyde/ethanol -0.20 (11)

FAD/FADH2 -0.06 (11, 14)

Dehydroascorbic acid/ascorbic acid 0.08 (11, 12)

Chromanoxyl radical of tocopherol/tocopherol 0.48 (13, 14)

O2/H2O 0.82 (14)

H2O2/O2ï- 0.87 (14)

NO+/NOï 1.21 (15)

H2O2/H2O 1.32 (14)

ONOO-/NO2ï 1.40 (15)

ïOH, H+/HïO 2.31 (15)

Inactivation of aldehyde dehydrogenase by nitroglycerin in the presence and... 1533

of the supplemented nonprotein-bound LA as aphysiological antioxidant and underline the prob-lem, overlooked until recently, related to the differ-ential effects of LA in vitro and in vivo (13, 14). Ourresearch paper is a modest contribution to the ongo-ing discussion on this issue.

In the present study, we investigated in vitrothe inactivation of yeast aldehyde dehydrogenase(ALDH) by nitroglycerin (glycerol trinitrate; GTN)in the presence and absence of LA and DHLA.Moreover, in vivo studies were performed to answerthe question whether LA administered jointly withGTN will affect the ALDH activity in the rat liver.

The genes encoding ALDHs were divided intoseveral families and subfamilies. It should be notedthat sequence comparison demonstrated that both

yeast and mammalian ALDH isozymes contained -SH groups that could form a wide variety of oxida-tion products (15).

EXPERIMENTAL

Reagents

In this study, the formulations Thiogamma andNitracor were used, which contain LA and GTN,respectively, as pharmacologically active sub-stances. Thiogamma was obtained from HexalÆ

AG, (Holzkirchen, Germany). Nitracor was pur-chased from LEK S.A. and Pliva KrakÛw S.A.(Poland). Purified yeast ALDH, as well as DHLA,EDTA, Folin-Ciocalteau reagent, 4-methylpyrazole,NAD, propionaldehyde and rotenone were provided

Scheme 1. Design of the experiment investigating yeast ALDH activity in vitro. Two series of enzyme mixtures were prepared. The finalconcentration of ALDH was 0.3 mg/mL in all experiments. The concentration of thiols (LA and DHLA) in the enzyme mixtures was 1mM


by Sigma-Aldrich Chemical Company ((PoznaÒ,Poland). All the other reagents were of analyticalgrade and were obtained from Polish ChemicalReagent Company (POCh, Gliwice, Poland).

Animals

Animal experiments were conducted in accor-dance with the guidelines for animal experiments ofAnimal Research Committee and were approved bythe Jagiellonian University Ethic Committee. Thestudy was carried out on male Wistar rats using themodified experimental design of Dudek et al. (16,17). The animals were divided randomly into threegroups of 8 animals each. The first group was treat-ed with 0.9% NaCl (0.6 mL, three times a day, s.c.)

for 8 days (control group). The second group wastreated with GTN (30 mg/kg b.w., s.c., divided intothree doses) for 8 days. The third group was treatedwith GTN and LA jointly for 8 days (GTN 30 mg/kgb.w., s.c., divided into three doses and LA 100mg/kg b.w., i.p., divided into two doses, respective-ly).

Animals were sacrificed on the 9th day by cer-vical dislocation and the livers were excised,washed in 0.9% NaCl, placed in liquid nitrogen andstored at -70OC until ALDH activity test was per-formed.

Methods

Preparation of liver homogenates

The frozen livers were weighed andhomogenates were prepared by homogenization of 1g of the tissue in 4 mL of 0.1 M phosphate buffer,pH 7.4 using an IKA-ULTRA-TURRAX T8homogenizer.

Determination of ALDH activity in the rat liver

homogenate

The assay mixture contained liver homogenate,sodium phosphate buffer (pH 8.2), NAD, EDTA, 4-methylpyrazole and rotenone. The reaction was initi-ated by the addition of propionaldehyde as a sub-strate. 4-Methylpyrazole was added to inhibit alcoholdehydrogenase, and rotenone to inhibit mitochondri-

Table 3. The effect of GTN administered in combination with LAfor 8 days on the ALDH activity in the rat liver.

TreatmentALDH

[U/mg protein]

Control group 45.28 ± 10.85

GTN group 21.12***± 3.87

GTN + LA group 26.25*** ± 5.38

***Differences significant at p < 0.001 in comparison to controlvalue. The activity of ALDH was calculated by using the molarextinction coefficient of reduced NAD of 6.22 mM-1 cm-1 at 340nm. Specific activity of the enzyme was expressed as nmolNADH/min (U)/mg-protein.

Figure 2. The LA and DHLA-induced restoration of GTN-inhibited ALDH activity in vitro. The ALDH activity was assayed with propi-onaldehyde (8 µM) as a substrate by measuring the reduction of NAD (1 mM) at 340 nm. Data are presented as a percentage relative tothe control (100%) (without GTN and thiols). Data are shown as the mean ± SD. Significant vs. control sample: ** p < 0.01; *** p < 0.001.Significant vs. GTN sample: # p < 0.05, ### p < 0.001


al NADH oxidase. The blank sample in which thehomogenate was omitted was run simultaneously.The activity of ALDH was calculated using themolar extinction coefficient of NADH of 6.22 mM-1cm-1 at 340 nm with the use of a modified pro-tocol published earlier (18, 19). Specific activity of theenzyme was expressed as nmol of NADH producedper 1 mg of protein per 1 min. The protein content wasmeasured using the method of Lowry et al. (20).

Determination of yeast ALDH activity

The final concentration of ALDH was 0.3mg/mL in all experiments. The concentration of thi-ols (LA and DHLA) in the enzyme mixtures was 1mM. Two series of enzyme mixtures were prepared(Scheme 1). All enzyme-containing mixtures wereincubated at a temperature of 25OC.

Series I was prepared as follows:

Enzyme mixture 1. ALDH was preincubatedwith 0.1 mM GTN in 50 mM sodium phosphatebuffer (pH 8) for 20 min.

Enzyme mixture 2. ALDH was preincubatedwith 0.1 mM GTN in 50 mM sodium phosphatebuffer (pH 8) for 5 min, then LA was added and themixture was incubated further for 15 min.

Enzyme mixture 3. ALDH was preincubatedwith 0.1 mM GTN in 50 mM sodium phosphate

buffer (pH 8) for 5 min, then DHLA was added andthe mixture was incubated further for 15 min.

Series II was prepared as follows:Enzyme mixture 1. ALDH was preincubated

with 0.1 mM of GTN in 50 mM sodium phosphatebuffer (pH 8) for 5 min.

Enzyme mixture 2. ALDH was preincubatedwith LA in 50 mM sodium phosphate buffer (pH 8)for 15 min, then 0.1 mM GTN was added and themixture was incubated further for 5 min.

Enzyme mixture 3. ALDH was preincubatedwith DHLA in 50 mM sodium phosphate buffer (pH8) for 15 min, then 0.1 mM GTN was added and themixture was incubated further for 5 min.

The assay mixture (1 mL) containing sodiumphosphate buffer (pH 8.2), propionaldehyde and NADwas pipetted into a cuvette. The reaction was initiatedby the addition of the indicated enzyme mixture to thecuvette and absorbance change at 340 nm was moni-tored for 2 min at 25OC to calculate the rate of NADHproduction and to compare it with the sample contain-ing only ALDH without GTN (control sample). Dataare presented as a percentage relative to control (100%).


All statistical calculations were carried outwith the STATISTICA 10.0 computer program

Figure 3. The LA and DHLA-induced prevention of ALDH inactivation by GTN in vitro. The ALDH activity was assayed with propi-onaldehyde (8 µM) as a substrate by measuring the reduction of NAD (1 mM) at 340 nm. Data are presented as a percentage relative tothe control (100%) (without GTN and thiols). Data are shown as the mean ± SD. Significant vs. control sample: *** p < 0.001. Significantvs. GTN sample: ## p < 0.01; ### p < 0.001.


using a one-way ANOVA followed by the Tukeypost-hoc test. Data from in vitro studies are present-ed as the mean ± SD of 3-4 independent experimentsin several repetitions. Data from in vivo experimentsare given as the mean ± SD for each group of ani-mals. For all data, the values of p < 0.05 were con-sidered to be statistically significant.

RESULTS

Activity of ALDH in the rat liver homogenatesAs shown in Table 3, the activity of ALDH in

the rat liver of the second group (GTN) and the thirdgroup (GTN + LA) was statistically significantlydecreased vs. control group (first group).

Activity of yeast-derived ALDH

The results obtained in series I are presented inFigure 2. The experiment indicated that 20-minincubation with GTN induced a decrease in theALDH activity to 8.2% (mixture I) as compared tothe control (100%). A five-minute preincubation ofthe enzyme with 0.1 mM GTN and then a 15-minincubation with 1 mM thiols: LA or DHLA (mixture2 and 3, respectively) caused a decrease in ALDHactivity to 31.6% and 71.9%, respectively, com-pared to the control (100%). This indicates that both1 mM thiols (LA and DHLA) were able to restoreALDH activity inhibited by GTN, but DHLA wasmore effective than LA.

The results obtained in series II are presentedin Figure 3. The experiment demonstrated that a 5-min incubation with GTN lowered ALDH activity to27% (mixture 1) compared to the control (100%).When the samples of ALDH were preincubated with1 mM LA or DHLA (mixture 2 and 3, respectively)for 15 min and then with 0.1 mM GTN for 5 min, theALDH activity changed to 68.1% and 106.9%,respectively, compared to the control (100%).

This indicates that both 1 mM thiols (LA andDHLA) were able to protect ALDH against theinhibitory effect of GTN, but DHLA was moreeffective than LA.

DISCUSSION AND CONCLUSIONS

In line with literature data, the present studiesdemonstrated that GTN inhibited ALDH activityboth in vivo and in vitro. Already in 1985, Towell etal. (21) observed an 88% inhibition of erythrocyticALDH in GTN-treated patients. Wenzel et al. (22)indicated that GTN was a potent inhibitor of bothpurified yeast ALDH as well as ALDH in isolatedheart mitochondria from in vivo GTN-treated rats.The next study of the same research group demon-strated that GTN inhibited ALDH activity in circu-lating white blood cells (WBCs) of healthy volun-teers (23). The studies of Beretta et al. (24) showedthat GTN blocked ALDH activity in isolated ratliver mitochondria, and also of recombinant humanALDH.

It is currently known that GTN is an inhibitorof ALDH (i.e., causes the loss of dehydrogenaseactivity) because it serves as a substrate for itsreductase activity. This means that ALDH catalyzesthe formation of glyceryl dinitrate (GDN) (1,2-GDN/1,3-GDN ~ 8 : 1) and nitrite (NO2

-) fromGTN. The electrons for this reduction are providedby cysteine-thiols at the active site of ALDH that areconverted to an intramolecular disulfide bond,which leads to a drop in the catalytic activity of theenzyme (24ñ26). A simplified scheme of thisprocess can be expressed as:

GTN + ALDH-(SH)2 (active) → 1,2-GDN + NO2-

+ H+ + ALDH-(SS)(inactive)

The present results obtained in vitro support therole of DHLA both in the restoration of ALDHactivity after inhibition by GTN and in the protec-tion of the enzyme against the inhibitory effect ofGTN. ALDH activity was not inhibited by GTN ifDHLA was added prior to GTN. Also, DHLA large-ly restored the inhibited ALDH activity if addedafter GTN. It was confirmed by studies of otherauthors. Namely, regeneration of the oxidized ñSHgroups of ALDH could be achieved via an exchangewith ñSH groups of DHLA (22, 24) what, in a sim-plified way, can be expressed as:

ALDH-(SS)(inactive) + DHLA-(SH2) →ALDH-(SH)2 (active) + LA-(SS)

Figure 4. Chemical structure of β-lipoic acid (β-LA). β-LA is abiological oxidation product of LA, which was proven in in vitro,but not in vivo studies (12, 27).


It is interesting and surprising that under in vitroconditions also LA prevented inactivation of theenzyme and had an impact on ALDH already inhibit-ed by GTN. Since this is the first study examining theeffect of GTN on the ALDH activity in the presenceof the oxidized form of DHLA, i.e. LA, this result isdifficult to explain because of too few data. In vitrostudies revealed that LA was capable of scavengingnumerous ROS and RNS, which indicated that despitebeing in its oxidized form LA behaved as a reducingagent (antioxidant). β-Lipoic acid (β-LA) (Fig. 4) is abiological oxidation product of LA, as demonstratedonly by in vitro studies (12, 27, 28). It means thatreduction process of the oxidized ñSH groups ofALDH, in a simplified form, can be expressed as:

ALDH-(SS)(inactive) + LA-(SS) + H2O →ALDH-(SH)2 (active) + β-LA

Is it possible? Further studies are necessary toverify this undoubtedly intriguing hypothesis.

On the other hand, the in vivo experimentsindicated that treatment of rats with LA and GTN incombination did not provide any protection againstGTN-induced ALDH inhibition. This is in contrastwith the results of Wenzel et al. (23), who reportedthat administration of a single sublingual GTN dosedecreased ALDH activity in WBCs in healthy vol-unteers and that these effects were prevented by oralLA pretreatment 15 min before GTN.

This difference could possibly be due to anumber of factors, including different experimentalconditions. In our opinion, the level of oxidativestress is the core of the problem since oxidativestress was undoubtedly much lower after GTNadministration at a single dose compared withchronic GTN treatment (8 days). The sulfhydrylgroups of cysteine residues (Cys-SH) in proteins (P-SH) can form a wide variety of oxidation products,including reversible oxidation states of disulfidebonds (P-SS) and sulfenic acid (P-SOH), and theirreversible higher oxidation states of sulfinic acid(P-SO2H) and sulfonic acid (P-SO3H) (15, 29).Thus, the plausible proposal is that under our exper-imental conditions in vivo the sulfhydryl groups ofcysteine residues at the active site of ALDH can beirreversible oxidized to sulfinic acid (P-SO2H) andsulfonic acid (P-SO3H) that cannot be reduced byLA and DHLA, but in experimental conditions invivo described in the above-mentioned paper byWenzel et al. (23) the activity of the enzymedecreased due to reversible oxidation of sulfhydrylgroups of cysteine residues in ALDH to disulfidebonds that can be reduced by LA and DHLA.

It should be mentioned that recent experi-mental and clinical studies indicate a beneficialeffect of exogenous LA in many diseases accom-panied by oxidative stress (30ñ36) and simultane-ously, the ability of LA and DHLA to function asphysiological antioxidants is questioned (13, 14).It means that the mechanism of pharmacologicalactions of LA in vivo still remains undiscovered.This issue definitely requires further studies themore so that searching for the mechanisms ofpharmacological actions of LA is important fromthe point of view of both basic research and itspractical applicability.

In summary, the obtained results seem to con-firm earlier reports indicating the differential effectsof LA in vitro and in vivo.


The authors do not have any conflict of interestregarding this manuscript.

Acknowledgments

The in vivo research was supported by statuto-ry funds of the Faculty of Medicine and Faculty ofPharmacy, Jagiellonian University, CollegiumMedicum, KrakÛw, Poland; the in vitro study wassupported by the National Science Centre, KrakÛw,Poland, grant No. 2011/01/B/NZ4/01675. The pub-lication of this paper was financially supported byLeading National Research Centre KNOW,KrakÛw, Poland.

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22. Wenzel P., Hink U., Oelze M., Schuppan S.,Schaeuble K. et al.: J Biol. Chem. 282, 792(2007).

23. Wenzel P., Schulz E., Gori T., Ostad M.A.,M‰thner F. et al.: J. Pharmacol. Exp. Ther. 330,63 (2009).

24. Beretta M., Sottler A., Schmidt K., Mayer B.,Gorren A.C.: J. Biol. Chem. 283, 30735 (2008).

25. Chen Z., Stamler J.S.: Trends Cardiovasc. Med.16, 259 (2006).

26. Chen Z., Zhang J., Stamler J.S.: Proc. Natl.Acad. Sci. USA 99, 8306 (2002).

27. Krishnan C.V., Garnett M.: Int. J. Electrochem.Sci. 6, 3607 (2011).

28. Trujillo M., Radi R.: Arch. Biochem. Biophys.397, 91 (2002).

29. Lee C.F., Paull T.T., Person M.D.: J. ProteomeRes. 12, 4302 (2013).

30. Papanas N., Ziegler D.: Expert Opin.Pharmacother. 15, 2721 (2014).

31. Iciek M., Marcykiewicz B., Bilska-Wilkosz A.,Soko≥owska-Jeøewicz M., K≥apciÒska J.:Pharmacol. Rep. 66, 325 (2014).

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Received: 15. 12. 2015


Biofilm is the microbial lifestyle in natural andman-made environments; it may be formed on awide variety of surfaces, including tissues orindwelling medical devices. This structure express-es the importance for public health as the result of itsrole in several infectious diseases as cystic fibrosis,native valve endocarditis, otitis media, periodonitisand chronic prostatitis, and especially in the infec-tions associated with biomaterials (e.g., catheters,prosthesis, etc.). It has to be known that biofilm isthe structure difficult to eradicate as the result ofembedding of the bacterial cells in self-producingslime ñ the mixture of polysaccharides and mineralssuch as calcium which are essential for the biofilmstructural integrity; slime protects the bacterial cellsagainst the activity of both immune system and anti-bacterial agents (1, 2).

The most important pathogens related withinfections associated with medical indwelling

devices are coagulase-negative staphylococci(CoNS) (3, 4). Among them, the most frequentlyisolated is S. epidermidis, followed by S. haemolyti-cus. S. haemolyticus may be also responsible forsuch infections as septicemia, peritonitis, nativevalve endocarditis, otitis media and urinary tractinfections. It is well known that S. haemolyticusvery often expresses multidrug resistance, includingmethicillin-resistance and even insensitivity to gly-copeptides, what makes this staphylococcal speciesthe most ìdangerousî among CoNS (5-7).

Linezolid, vancomycin, tigecycline and dapto-mycin belong to group of antistaphylococcal drugspossessing activity against methicillin-resistant andmulti-drug resistant isolates. The aim of this studywas to asses the influence in vitro of the above men-tioned antibiotics on biofilm formation by S.haemolyticus and eradication of the mature struc-ture.

ANTI-ADHESIVE AND ANTI-BIOFILM ACTIVITIES IN VITRO OF LINEZOLID, VANCOMYCIN, TIGECYCLINE AND DAPTOMYCIN AGAINST

STAPHYLOCOCCUS HAEMOLYTICUS

MAREK JUDA*, PAWE£ HELON and ANNA MALM

Department of Pharmaceutical Microbiology, Medical University of Lublin, 1 Chodüki St., 20-093 Lublin, Poland

Abstract: Biofilm may be formed on wide variety of surfaces, including indwelling medical devices, leadingto several infectious diseases, e.g., bacteremia and sepsis. The most important pathogens related with infectionsassociated with medical devices are coagulase-negative staphylococci, including Staphylococcus haemolyticusñ bacterial species which express quite often the multidrug resistance. The four clinical multiresistant andmethicillin-resistant S. haemolyticus were included in the present study. The evaluation of drug susceptibilitywas performed by using disc-diffusion method and broth microdilution method according to EuropeanCommittee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The biofilm formation on theNelaton catheter and the effect of linezolid, vancomycin, tigecycline and daptomycin on the biofilm formationand disruption of mature structure was based on the method with TTC (2,3,5-triphenyltetrazolium chloride).The adhesion process of S. haemolyticus to the Nelaton catheter was inhibited by antibiotics, as follows: line-zolid at concentration 0.25-0.5 ◊ MIC, vancomycin ñ concentration 0.5 ◊ MIC, tigecycline ñ concentration0.25-4 ◊ MIC and daptomycin ñ concentration 0.06-1 ◊ MIC, depending on the isolate. Linezolid inhibited thebiofilm formation at concentration between 0.5-1 ◊ MIC, vancomycin ñ 1-2 ◊ MIC, tigecycline ñ 0.5-4 ◊ MICand daptomycin ñ 0.06-2 ◊ MIC. The concentration of linezolid eradicating the mature biofilm was found to be1-2 ◊ MIC, vancomycin ñ 2-8 ◊ MIC, tigecycline - 2-4 ◊ MIC and daptomycin ñ 0.06-2 ◊ MIC. The most activeantibiotic against S. haemolyticus biofilm formation and disruption of mature structure seems to be daptomycin.

Keywords: biofilm, Staphylococcus haemolyticus, adhesion, disruption, antibiotics

1539

* Corresponding author: e-mail: [email protected]; phone/fax:+48-81-4487100

1540 MAREK JUDA et al.

EXPERIMENTAL

Bacterial strains

The four clinical multiresistant isolates ofmethicillin-resistant S. haemolyticus were includedin the present study. The strains were stored in tryp-ticasein soy broth (TSB) with 50% glycerol in -72OC.

Determination of drug susceptibility

The evaluation of susceptibility of S.haemolyticus to antimicrobial agents was performedaccording to European Committee on AntimicrobialSusceptibility Testing (EUCAST) guidelines. Thedisc diffusion method was used in evaluation of sus-ceptibility to the following antibiotics: cefoxitin,erythromycin, clindamycin, chloramphenicol, tetra-cycline, ciprofloksacin, rifampicin, gentamicin, sul-famethoxazole/trimethoprim, fusidic acid.

Isolation of bacterial DNA

The DNA Genomic Mini Kit (A&ABiotechnology) was used in isolation of S. epider-midis DNA according to the manufacturer guidelines.

Identification of mecA by PCR

The following sequence of the primer for mecAwas used: 5í-AAAATCGATGGTAAAGGTTGGC-3í (forward primer), 5í-AGTTCTGCAGTACCG-GATTTGC-3í (reverse primer). For the reaction ofPCR the PCR REDTaqÆ Ready MixTM PCR Mixwith MgCl2 (Sigma-Aldrich) was used. The finalmixture volume of PCR reaction was 25 mL andcontained 12.5 mL of REDTaq Ready Mix, 1 mL ofeach forward and reverse primer (concentrationbetween 0.1-1.0 mM), 1 mL of DNA (50-200 ng)and 9 mL of water. The reaction was performed byusing Whatman Biometra thermocycler, while thePCR products were subjected to agarose gel elec-trophoresis (2% agarose, 1 ◊ TRIS-acetate-EDTA,120 mV, 40 min). The conditions of 35 cycles of thePCR reaction was following: 94OC ñ 1 min, 55OC ñ1 min, 72OC ñ 1 min. The gel was stained with ethid-ium bromide and PCR product for mecA (533 bp)was visualized using Wilbert Lambert transillumi-nator by comparison with a molecular size marker ñGene RulerTM 100bp DNA Ladder (Fermentas).

Determination of the minimal inhibitory concen-

tration (MIC) and minimal bactericidal concen-

tration (MBC) to the linezolid, vancomycin, tige-

cycline and daptomycin

The MIC of antibiotics for S. haemolyticus iso-lates was performed by the broth microdilution

method, using two-fold dilutions of antibiotic inMueller-Hinton broth (MHB) or MHB supplement-ed with calcium for estimation of susceptibility todaptomycin. Inoculum of bacterial strains was 5 ◊105 colony forming units (CFU) per 1 mL. Afterincubation at 35OC for 18 h, the MICs were assessedvisually as the lowest antibiotic concentration show-ing complete bacterial growth inhibition. In order todetermine MBC, 10 mL of the bacterial culture fromeach well that showed thorough growth inhibition,from the last positive one and from the growth con-trol was streaked onto trypticasein soy agar (TSA).After incubation at 35OC for 24 h, the MBCs wereassessed visually as the lowest antibiotic concentra-tions at which there was no bacterial growth.

Biomaterials

All assays were carried out on the Nelatoncatheter made of polichloride vinyl. The catheterwas cut aseptically into ca 0.5 cm2 fragments andplaced into Petri dishes.

Determination of S. haemolyticus adhesion to the

Nelaton catheter in vitroThe ability of adhesion of the S. haemolyticus

isolates to the catheter was studied in vitro by TTC(2,3,5-triphenyltetrazolium chloride) method basedon the ability of living cells to reduce tetrazoliumsalt to red formazan precipitates (5). The standard-ized bacterial suspensions (0.5 according toMcFarland standard) in sterile PBS (phosphate-buffered saline) were incubated with the catheter for2 h at 35-37OC. Nonadherent cells were removed bycareful rinsing of catheter fragment with sterile PBSand then resuspended in MHB with one drop of 1%TTC, followed by overnight incubation at 35-37OC.Adhesion process was determined visually by anappearance of red formazan precipitates, both on thecatheter surface and in the medium.

Determination of biofilm formation by S.haemolyticus on the Nelaton catheter in vitro

The ability of formation of biofilm by the S.haemolyticus isolates on the catheter was studied invitro also by TTC method. The standardized bacter-ial suspensions (0.5 according to McFarland stan-dard) in MHB were incubated with appropriate bio-material for 24 h at 35-37OC. Nonadherent cellswere removed by careful rinsing of the catheter frag-ment with sterile PBS and then resuspended in freshMHB. Medium changing and catheter washing pro-cedures after overnight incubation at 35-37OC wererepeated three times. Finally, one drop of 1% TTCsolution was added, followed by overnight incuba-

Anti-adhesive and anti-biofilm activities in vitro of... 1541

tion at 35-37OC. Biofilm formation was determinedvisually on the basis of an appearance of red for-mazan precipitates both on the catheter surface andin the medium.

The effect of antibiotics on adhesion and biofilm

formation by S. haemolyticus on the Nelaton

catheter in vitroThis assay was based on the TTC method

described above. In each experiment, several con-centrations of antibiotics were used as multiplicationof MIC: 0.5; 1.0; 2.0; 4.0; 8.0; 16.0; 32.0; 64.0 ◊MIC. (I) In order to assay the effect of antibiotics onadhesion, the standardized bacterial suspensions insterile PBS containing appropriate antibiotic wereincubated with the catheter at 35-37OC. Then, a dropof 1% TTC solution was added, followed byovernight incubation at 35-37OC. The minimal con-centration of antibiotic inhibited adhesion processwas determined visually as the concentration wherethe red formazan precipitates were not found, bothon the catheter surface and in the medium. (II) Inorder to assay the effect of antibiotic on biofilm for-mation, the bacterial suspensions in TSB containingantibiotic were incubated with appropriate biomate-rial for 72 h at 35-37OC, with medium changing andcatheter washing process as described above. Then,a drop of 1% TTC solution was added, followed byovernight incubation at 35-37OC. The minimal con-centration of antibiotic inhibited biofilm formation

was determined visually as the concentration wherethe red formazan precipitates were not found, bothon the catheter surface and in the medium. (III) Inorder to assay the effect of antibiotic on biofilmeradication, the mature 72-h biofilms were incubat-ed in the presence of antibiotic for 24-h and then adrop of 1% TTC solution was added, followed byovernight incubation at 35-37OC. The minimal con-centration of antibiotic eradicated the maturebiofilm was determined visually as the concentra-tion where the red formazan precipitates were notfound, both on the catheter surface and in the medi-um.

Reproducibility of the results: All results weredone in triplicate. Representative data are presented.

RESULTS

On the basis of disc-diffusion method the drug-resistance patterns of S. haemolyticus strains used inthe present study were evaluated (Table 1). Allstrains possessed the mecA gene.

As shown in Table 2, on the basis of MIC thestudied strains were susceptible to linezolid (LZD),vancomycin (VA), tigecycline (TGC) and dapto-mycin (DA). The MBC/MIC ratio showed the bac-tericidal effect for vancomycin and daptomycin orbacteriostatic effect for linezolid and tigecycline.

The power of anti-adherent and anti-biofilmactivity of the antibiotics was assessed on the basis

Table 2. The minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) andMBC/MIC ratio of linezolid, vancomycin, tigecycline and daptomycin for Staphylococcus haemolyticus.

Antibiotic MIC (mg/L) MBC (mg/L) MBC/MIC

LZD 1 8-48 8-48

VA 2 2-8 1-4

TGC 0.06-0.25 2-8 8-64

DA 0.5-1 1-4 2-4

LZD - linezolid, VA - vancomycin, TGC - tigecycline, DA - daptomycin.

Table 1. The drug resistance patterns of Staphylococcus haemolyticus.

Isolate Drug resistance pattern

1 FOX, E, CC, CIP, GM, SXT

2 FOX, CIP, RA, GM

3 FOX, E, CC, TE, C, CIP, GM, SXT

4 FOX, E, CC, GM

Legend: FOX - cefoxitin, E - erythromycin, CC - clindamycin, TE - tetracycline, RA - rifampicin, CIP -ciprofloxacin, C - chloramphenicol, GM - gentamicin, SXT - sulfamethoxazole/trimethoprim.

1542 MAREK JUDA et al.

of the ratio between MIC values and minimal con-centration of a given antibiotic inhibiting the biofilmformation or eradicating the mature structure.According to Table 3, the adhesion process of S.haemolyticus to the Nelaton catheter was inhibitedby antibiotics, as follows: linezolid ñ 0.25-0.5 ◊MIC, vancomycin ñ 0.5 ◊ MIC, tigecycline ñ 0.25-4◊ MIC and daptomycin ñ 0.06-1 ◊ MIC, dependingon the isolate. The somewhat higher concentrationsof antibiotics were necessary for the inhibition ofbiofilm formation; linezolid inhibited this process atconcentration between 0.5-1 ◊ MIC, vancomycin ñ1-2 ◊ MIC, tigecycline ñ 0.5-4 ◊ MIC and dapto-mycin ñ 0.06-2 ◊ MIC. The concentration of line-zolid eradicating the mature biofilm was found to be1-2 ◊ MIC, vancomycin ñ 2-8 ◊ MIC, tigecycline ñ2-4 ◊ MIC and daptomycin ñ 0.06-2 ◊ MIC.


Nowadays, the biofilm associated infectionsare one of the most important problems in medicine.The 65% of nosocomial infections are assessed to berelated with biofilm formation. The many methodshave been studied to inhibits the biofilm formationor even disruption of the mature structure (1, 2, 4).One of the method used in prevention of biofilm for-mation is the incorporation of the antibiotic or non-antibiotic agents into biomaterials. According to theliterature data (8-10) some antibiotics: imipenem,being an inhibitor of cell wall synthesis, or the

inhibitors of bacterial protein synthesis as aminogly-cosides, fluoroquinolones, oxazolidinones, strep-togramins, have been found to influence on thebiofilm formation by several bacterial species.Moreover, also the non-antibiotic chemical com-pounds are promising agents inhibiting the forma-tion of biofilm structure, e.g., EDTA, usnic acid,surfactin or ovotransferin (10-12).

As presented here, linezolid (oxazolidinone),vancomycin (glycopeptide), tigecycline (glycylcy-cline) and daptomycin (lipopeptide) affected varioussteps of biofilm development by methicillin resistantS. haemolyticus, i.e., adhesion, formation and eradi-cation of mature biofilm structure formed. It is gen-erally accepted, that these antibiotics are very oftenthe ìdrug of choiceî in the treatment of infectionscaused by multidrug resistant staphylococci, espe-cially S. aureus.

It was found that the above drugs are able toprevent the adhesion of S. haemolyticus cells.However, the higher concentrations were necessaryin inhibition of biofilm formation or in disruption ofmature structure. In some of the cases these concen-trations were higher than MIC values estimatedagainst the planktonic cells. It was found that line-zolid and daptomycin expressed the best anti-biofilm properties, both in formation and in disrup-tion of the mature structure. These agents were ableto eradicate of S. haemolyticus biofilm even in thesubinhibitory concentrations in opportunity to van-comycin and tigecycline. However, it has to be

Table 3. The influence of linezolid, vancomycin, tigecycline and daptomycin on adhesion, biofilm formation and eradication of maturestructure of Staphylococcus haemolyticus.

Minimal Multiplication Antibiotic concentration of of

antibiotic (mg/L) MIC values

Adhesion 0.25-0.5 0.25-0.5

LZD Biofilm formation 0.5-1 0.5-1

Eradication of biofilm 1-2 1-2

Adhesion 1 0.5

VA Biofilm formation 2-4 1-2

Eradication of biofilm 4-16 2-8

Adhesion 0.03-0.125 0.125-2

TGC Biofilm formation 0.125--0.25 0.5-4

Eradication of biofilm 0.125-1 2-4

Adhesion 0.03-1 0.06-1

DA Biofilm formation 0.03-1 0.06-2

Eradication of biofilm 0.03-2 0.06-2

LZD - linezolid, VA - vancomycin, TGC - tigecycline, DA - daptomycin.

Anti-adhesive and anti-biofilm activities in vitro of... 1543

remembered that linezolid expresses bacteriostaticeffect ñ its inhibits only the growth of the bacterialcell but does not kill the population, in contrast todaptomycin (being a bactericidal drug).

It was also found that vancomycin requireshigher concentrations both in biofilm formation andin eradication of the structure. This observation con-firms that antibiotics inhibiting the cell wall synthe-sis are less active against biofilm formation in oppo-site to antibiotics inhibiting the protein synthesis.The previous data showed that even antibioticswhich do not express the antibacterial activityagainst the species can decrease the ability ofbiofilm formation, e.g., azithromycin againstbiofilm of Pseudomonas aeruginosa as the result ofinhibition of the alginate synthesis (13). However, ithas to be noted that the addition of the other chemi-cal compounds can increase the anti-biofilm activityof antibiotics, e.g., vancomycin + heparin orminocycline + EDTA (10, 14).

The mechanism of influence of antibiotics onbiofilm formation is still unknown. However, it hasbeen found that some of the antibiotics(dicloxacillin) decreased the synthesis of extracellu-lar molecule - poly-N-acetylglucosamine (PNAG)and changed the hydrophobicity of bacterial cell sur-face of S. epidermidis (15). In opposite, Rachid et al.(16) showed that tetracyclines increased the expres-sion of ica operon involved in synthesis of polysac-charide intracellular adhesine (PIA). Recently, acapsular polysaccharide (CP) was proposed to be animportant virulence factor of S. haemolyticus.However, the presence of ica operon has beenreported but to date its contribution in these speciesto biofilm formation is unclear (17).

As it was mentioned above, one of the factorsresponsible for the low activity of antibiotics againstthe bacterial strains embedded in biofilm is slowpenetration of the drugs into the structure. The dataof Stewart et al. (18) showed that daptomycin canreadily penetrate thick S. epidermidis biofilms inminute or two what is much shorter that the durationof antibiotic exposure which is typically tens ofhours.

Daptomycin effected the processes involved inthe biofilm formation and caused disruption of themature structure at comparable concentrations. Themost active antibiotic against S. haemolyticusbiofilm formation and disruption of mature structureseems to be daptomycin.

REFERENCES

1. Donlan R.M.: Emerg. Infect. Dis. 8, 881 (2002).2. Costerton J.W., Stewart P.S., Greenberg E.P.:

Science 284, 1318 (1999).3. Agarwal A., Singh K.P., Jain A.: FEMS

Immunol. Med. Microbiol. 58, 147 (2010).4. Gˆtz F.: Mol. Microbiol. 43, 1367 (2002).5. Gallimore B., Gagnon R.F., Subang R.,

Richards G.K.: J. Infect. Dis. 164, 1220 (1991).6. Fredheim E.G.A., Klingenberg C., Rohde H.,

Frankenberg S., Gaustad P. et al.: J. Clin.Microbiol. 47, 1172 (2009).

7. Silva P.V., Cruz R.S., Keim L.S., Paula G.R.,Carvalho B.T.F. et al.: Mem. Inst. OswaldoCruz 108, 812 (2013).

8. Gander S., Hayward K., Finch R.: J.Antimicrob. Chemother. 49, 301 (2002).

9. Raad I., Hanna H., Ijang Y., Dvork T., ReitzelR. et al.: Antimicrob. Agents Chemother. 51,1656 (2007).

10. Raad I., Hanna H., Dvorak T., Chaiban G.,Hachem R.: Antimicrob. Agents Chemother.51, 78 (2007).

11. Percival S.L., Kite P., Eastwood K., Murga R.,Carr J. et al.: Infect. Control Hosp. Epidemiol.26, 515 (2005).

12. Juda M., Paprota K., Ja≥oza D., Malm A.,Rybojad P.: Ann. Agric. Environ. Med. 15, 237(2008).

13. Yakandawala N., Gawande P.V., LoVetri K.,Madhyastha S.: J. Appl. Microbiol. 102, 722(2007).

14. Hoffmann N., Lee B., Hentzer M., RasmussenT.B., Song Z.: Antimicrob. Agents Chemother.51, 3677 (2007).

15. Raad I., Chatzinikolaou I., Chaiban G., HannaH., Hachem R. et al.: Antimicrob. AgentsChemother. 47, 3580 (2003).

16. Cerca N., Martins S., Sillankorva S., JeffersonK.K., Pier G.B. et al.: Appl. Environ. Microbiol.71, 8677 (2005).

17. Rachid S., Olsen K., Witte W., Hacker J.,Ziebuhr W.: Antimicrob. Agents Chemother.44, 3357 (2000).

18. Flahaut S., Vinogradov E., Kelley K.A.,Brennan S., Hiramatsu K., Lee J.C.: J.Bacteriol. 190, 1649 (2008).

19. Stewart P.S., Davison W.M., Steenbergen J.N.:Antimicrob. Agents Chemother. 53, 3505(2009).

Received: 12. 01. 2016


The concept of multi-target drugs has arisenstudying molecular mechanism of action amongseveral efficient drugs, such as neuroleptics, antide-pressants, and antineurodegenerative agents whichaffect many targets simultaneously. Moreover, thepattern of promiscuous drugs is based on the factthat common central nervous disorders, such asdepression, schizophrenia, Alzheimerís or Parkin-sonís diseases, and epilepsy, tend to result frommultiple molecular abnormalities, and not from asingle defect. This multi-target strategy has expand-ed tremendously the number of potential targets andhas led to the introduction of new classes of drugswith potentially less serious side-effects and lowertoxicity (1, 2).

An arylpiperazine moiety is one of the mostuniversal templates used for designing agents activeat G-protein coupled receptors (GPCRs). Simplearylpiperazines are classified as non-selective recep-tor ligands, but long-chain arylpiperazines (LCAPs)

have been found to be serotonin receptor ligands, inparticular 5-HT1A and 5-HT2A. Their general chemi-cal structure contains an alkyl chain (2ñ4 methyleneunits) attached to the N4 atom of the piperazine moi-ety, and a terminal fragment: an amide or imide.Numerous studies have indicated that even a minorstructural modification within the LCAP ring or atthe terminal fragment (an amide or imide moiety)strongly affects receptor affinity and selectivity(3ñ7). For several years, we have been developingLCAP-class agents completed with an amide ring,which were evaluated in functional in vivo modelsof anxiety and depression (8ñ11).

Imidazolidine-2,4-dione is an important coreunit that exhibits a range of central and peripheralbiological activities and is incorporated into manydrugs with numerous therapeutic applications (12,13). Therefore, well-known antiepileptic agentssuch as phenytoin, mephenythoin, norantoin,methetoin, ethotoin, fosphenytoin are based on the

DRUG SYNTHESIS

NEW SPIROHYDANTOIN DERIVATIVES ñ SYNTHESIS, PHARMACOLOGICAL EVALUATION, AND MOLECULAR

MODELING STUDY

ANNA CZOPEK1*, AGNIESZKA ZAG”RSKA1, MARCIN KO£ACZKOWSKI1, ADAM BUCKI1,BEATA GRYZ£O1, JOANNA RYCHTYK1, MACIEJ PAW£OWSKI1, AGATA SIWEK2,

GRZEGORZ SATA£A3, ANDRZEJ BOJARSKI3, MONIKA KUBACKA4 and BARBARA FILIPEK4

1Department of Pharmaceutical Chemistry, 2Department of Pharmacobiology,4Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College,

Medyczna 9, 30-688 KrakÛw, Poland3Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences,

SmÍtna12, 31-343 KrakÛw, Poland

Abstract: A series of new arylpiperazinylpropyl derivatives of 8/6-phenyl-1,3-diazaspiro[4.5]decan-2,4-dioneand spiro[imidazolidine-4,1í-indene/naphthalene]-2,5-dione was synthesized and their affinity was evaluatedtoward serotonin 5-HT1A, 5-HT2A, 5-HT7 receptors, dopaminergic D2, D3 receptors, adrenergic α1 receptors, andserotonin transporter (SERT). The highest affinity for serotonin 5-HT1A/2A/7 receptors was found for compoundscontaining a tetralin or indane moiety in the imide part. Among these, two compounds (19, 20) were selectedfor further pharmacological in vivo studies. A binding mode of representative molecule 19, which behaved asa 5-HT1A agonist and weak 5-HT7 antagonist in the site of 5-HT1A/7, was also analyzed in computational stud-ies. Moreover, two highly selective (9 and 11) 5-HT2A receptor antagonists were obtained.

Keywords: imidazolidine-2,4-dione, long-chain arylpiperazines, multi-receptor ligands, spirohydantoin

1545

* Corresponding author: e-mail: [email protected]; phone: +48 12 6205450, fax: +48 12 6205450

1546 ANNA CZOPEK et al.

structure of the imidazolidine-2,4-dione (13).Moreover, imidazolidine-2,4-dione derivatives canalso be found as antiarrhythmics (azimilide), antimi-crobial drugs (nitrofurantoin), skeletal muscle relax-ants (dantrolene) and non-steroidal antiandrogens(nilutamide), while allantoin (5-ureidohydantoin) isused as a keratolytic, astringent, wound remedy,antacid and antipsoriatic drug (13). Imidazolidine-2,4-dione (hydantoin) can be substituted in severalpositions and the search for hydantoin-based drugsis ongoing.

In order to obtain compounds acting on multi-ple biological targets, two pharmacophoric systems(hydantoin and LCAPs) were combined. Followingthe results of our previous study (9, 11), we extend-ed our studies aimed at verification of the impact ofthe linker between spirohydantoin derivatives andthe arylpiperazine moiety. The influence on sero-tonin and dopamine receptor activity of differentìspiroî substituents at the 5 position of a hydantoinmoiety was studied. For this reason, we proposed tointroduce an aromatic ring into the ìspiroî sub-stituent as a flexible (9ñ16) or rigid (17ñ26) frag-ment. Furthermore, the arylpiperazine fragment waschanged into a 1,2,3,4,-tetrahydroisoquinoline moi-ety to diversify the affinity of the designed com-pounds for serotonin receptors.

In this paper, we report on the synthesis of newpropyl spirohydantoin derivatives and their biologi-cal evaluation toward monoaminergic receptors (α1,5-HT1A, 5-HT2A, 5-HT6, 5-HT7, D2, D3) and a sero-tonin transporter (SERT). We also discuss whetherthe proposed modifications will improve affinity forserotonin and dopamine receptors as well as allowsuitable multi-receptor profile characteristics forantidepressant or antipsychotic activity to beachieved. Furthermore, the interactions of com-pound 19 with 5-HT1A/7 receptors are discussedbased on molecular modeling study results.

EXPERIMENTAL

Chemistry

The structure of the final compounds 9-26 wasestablished on the basis of the results of elemental(C, H, N) and spectral (1H NMR, 19F NMR) analyses.NMR spectra were recorded on Varian Mercury 300MHz spectrometer (Varian Inc., Palo Alto, CA,USA); chemical shifts are expressed in parts permillion (ppm), using the solvent (CDCl3 or DMSO-d6) signal as an internal standard. Signal multipletsare represented by the following abbreviations: s(singlet), br s (broad singlet), d (doublet), t (triplet),m (multiplet). Melting points were determined in

open capillaries on an Electrothermal 9300 appara-tus and were uncorrected. Thin-layer chromatogra-phy (TLC) was performed on Merck silica gel 60F254 aluminium sheets (Merck; Darmstadt, Germa-ny), using the following mixtures of solvents: (S1)benzene/ethyl acetate/acetone (10 : 5 : 1, v/v/v) and(S2) acetone/isopropanol/chloroform (20 : 10 : 1,v/v/v). Elemental analyses for C, H, N were carriedon an Elementar Vario EL III apparatus (Hanau,Germany). LC/MS analysis was performed onWaters Acquity TQD system, with a Waters TQDquadrupole mass spectrometer with detection by UV(DAD) using an Acquity UPLC BEH C18 column(1.7 µm, 2.1 mm ◊ 100 mm). Water/acetonitrile gra-dient with 0.1% TFA was used as a mobile phase ata flow rate of 0.3 mL/min.

The starting spirohydantoins (1ñ4) and inter-mediate (7, 8) were prepared according to previous-ly described methods (9, 11).

1-(3-Chloropropyl)-8-phenyl-1,3-diazaspiro [4.5]

decan-2,4-dione (5)

The free base was obtained in 72% yield aswhite powder ; m.p. 212ñ214OC; TLC: Rf = 0.56(S1); Analysis: calcd. for C17H21N2O2Cl: C 63.64, H6.60, N 8.73%; found: C 63.63, H 6.73, N 8.50%.

1-(3-Chloropropyl)-6-phenyl-1,3-diazaspiro[4.5]

decan-2,4-dione (6)

The free base was obtained in 73% yield aswhite powder: yield 73%; m.p. 210ñ211OC; TLC: Rf = 0.64 (S1); Analysis: calcd. for C17H21N2O2Cl: C63.64, H 6.60, N 8.73%; found: C 63.66, H 6.65, N8.55%.

General procedure for preparing final compounds

9-26

An intermediate 1-(3-chloropropyl)-spirohy-dantoin (5 mmol) and the substituted 1-phenylpiper-azine or tetrahydroisoquinoline (10 mmol) in ethyl(9ñ12, 14, 15, 17ñ26) or butyl (13) alcohol or 2-methoxyethanol (16) were refluxed for 40 h sepa-rately. After cooling, the solvent was evaporated andthe residue was extracted with CHCl3 (3 ◊ 15 mL).The combined organic phases were dried, filteredoff and evaporated. The obtained oily product waspurified either by crystallization from anhydrousethanol (comp. 14, 15, 17ñ26) or by column chro-matography (comp. 9ñ13, 16), using a mixture ofsolvents acetone/isopropanol/chloroform (20 : 10 :1, v/v/v).

3-[3-(4-Phenylpiperazin-1-yl)propyl]-8-phenyl-

1,3-diazaspiro[4.5]decan-2,4-dione (9)

New spirohydantoin derivatives - synthesis, pharmacological... 1547

The free base was obtained in 59% yield aswhite powder; m.p. 188ñ190OC; 1H NMR (300 Hz,CDCl3, δ, ppm): 8.38 (s, 1H), 7.20ñ7.37 (m, 7H),6.83ñ6.99 (m, 3H), 3.64ñ3.68 (t, 2H, J = 7.20 Hz),3.17ñ3.20 (t, 4H, J = 4.70 Hz), 2.60ñ2.69 (m, 5H),2.46ñ2.51 (t, 2H, J = 7.20 Hz), 1.89ñ2.08 (m, 5H),1.70ñ1.83 (m, 5H). TLC: Rf = 0.07 (S1); 0.74 (S2);HPLC: Rt = 2.41 (99%); LC/MS (m/z): 447.6 [M +H]+. Analysis: calcd. for C27H34N4O2 ◊ H2O: C69.80, H 7.81, N 12.06%; found: C 69.68, H 7.43, N11.98%.

3-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]prop-

yl}-8-phenyl-1,3-diazaspiro[4.5]decan-2,4-dione

(10)

The free base was obtained in 67% yield aswhite powder; m.p. 179ñ181OC; 1H NMR (300 Hz,CDCl3 δ, ppm): 7.65 (br s, 1H), 7.20ñ7.33 (m, 5H),6.83ñ6.99 (m, 4H), 3.85 (s, 3H), 3.61ñ3.66 (t, 2H, J= 7.18 Hz), 3.07 (br s, 4H), 2.63 (br s, 5H), 2.44ñ2.48(t, 2H, J = 7.18 Hz), 1.93ñ2.08 (m, 5H), 1.65ñ1.90(m, 5H). TLC: Rf = 0.79 (S2); HPLC: Rt = 2.43(99%). Analysis: calcd. for C28H36N4O3: C 70.56, H7.61, N 11.76%; found: C 70.42, H 7.65, N 11.55%.

3-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-

8-phenyl-1,3-diazaspiro[4.5]decan-2,4-dione (11)

The free base was obtained in 55% yield aswhite powder; m.p. 197ñ199OC; 1H NMR (300 Hz,CDCl3 δ, ppm): 8.15 (s, 1H), 7.12ñ7.33 (m, 6H),6.73ñ6.83 (m, 3H), 3.63ñ3.67 (t, 2H, J = 7.00 Hz),3.20 (br s, 4H), 2.67ñ2.69 (m, 5H), 2.15ñ2.20 (t, 2H,J = 6.90 Hz), 1.97ñ2.08 (m, 5H), 1.67ñ1.81 (m, 5H).TLC: Rf = 0.71 (S2); HPLC: Rt = 2.64 (96%).Analysis: calcd. for C27H33N4O2Cl: C 67.42, H 6.91,N 11.65%; found: C 67.24, H 7.06, N 11.49%.

3-{3-[4-(3-Trifluoromethylphenyl)piperazin-1-

yl]propyl}-8-phenyl-1,3-diazaspiro[4.5]decan-

2,4-dione (12)

The free base was obtained in 69% yield aswhite powder; m.p. 214ñ216OC; 19F NMR (300 Hz,CDCl3 δ, ppm): -62.72 (s, 3F); 1H NMR (300 Hz,CDCl3, δ, ppm ): 8.54 (s, 1H), 7.18ñ7.36 (m, 6H),6.99ñ7.06 (m, 3H), 3.63ñ3.67 (t, 2H, J = 7.30 Hz),3.18ñ3.21 (t, 4H, J = 4.80 Hz), 2.56ñ2.68 (m, 5H),2.44ñ2.49 (t, 2H, J = 7.30 Hz), 1.85ñ2.06 (m, 6H),1.72ñ1.79 (m, 4H). TLC: Rf = 0.73 (S2); HPLC: Rt =2.76 (97%); LC/MS (m/z) 515.5 [M + H]+.

3-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)propyl]-8-

phenyl-1,3-diazaspiro[4.5]decan-2,4-dione (13)

The free base was obtained in 54% yield ascreamy powder; m.p. 179ñ181OC; 1H NMR (300 Hz,

CDCl3 δ, ppm): 1.60ñ1.72 (m, 4H), 1.91ñ2.02 (m,6H), 2.56ñ2.62 (m, 3H), 2.67ñ2.71 (t, 2H, J = 5.20Hz), 2.85ñ2.89 (t, 2H, J = 5.20 Hz), 3.59 (s, 2H),3.65ñ3.69 (t, 2H, J = 7.00 Hz), 6.96ñ7.32 (m, 9H),7.80 (s, 1H). TLC: Rf = 0.64 (S2); HPLC: Rt = 2.38(99%). Analysis: calcd. for C26H31N3O2: C 74.79, H7.48, N 10.06%; found: C 75.00, H 7.48, N 9.98%.

3-[3-(4-Phenylpiperazin-1-yl)propyl]-6-phenyl-

1,3-diazaspiro[4.5]decan-2,4-dione (14)

The free base was obtained in 60% yield aswhite powder; m.p. 208ñ209OC; 1H NMR (300 Hz,CDCl3 δ, ppm): 1.14ñ1.28 (m, 2H) 1.42ñ1.58 (m,3H), 1.76ñ1.91 (m, 5H), 2.04ñ2.09 (t, 2H, J = 7.00Hz), 2.44ñ2.47 (t 4H, J = 5.00 Hz,), 3.07ñ3.09 (m,1H), 3.14ñ3.18 (t, 4H, J = 5.00 Hz), 3.21ñ3.26 (t,2H, J = 7.00 Hz), 6.81ñ7.10 (m, 3H), 7.17ñ7.28 (m,8H). TLC: Rf = 0.09 (S1); 0.84 (S2); HPLC: Rt = 2.27(97%); LC/MS (m/z) 447.6 [M+H]+. Analysis:calcd. for C27H34N4O2: C 72.62, H 7.67, N 12.55%;found: C 72.45, H 7.84, N 12.55%.

3-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]prop-

yl}-6-phenyl-1,3-diazaspiro[4.5]decan-2,4-dione

(15)

The free base was obtained in 60% yield aswhite powder; m.p. 183ñ184OC; 1H NMR (300 Hz,CDCl3 δ, ppm): 7.16ñ7.27 (m, 7H), 6.83ñ7.02 (m,3H), 3.85 (s, 3H), 3.20ñ3.25 (t, 2H, J = 6.80 Hz),3.11ñ3.13 (m, 1H), 3.05 (br s, 4H), 2.51 (br s, 4H),2.08ñ2.12 (t, 2H, J = 7.20 Hz), 1.76ñ1.95 (m, 5H),1.39ñ1.57 (m, 3H), 1.14ñ1.26 (m, 2H). TLC: Rf =0.13 (S1), 0.77 (S2); HPLC: Rt = 2.25 (98%).Analysis: calcd. for C28H36N4O3: C 70.56, H 7.61, N11.76%; found: C 70.31, H 7.58, N 11.51%.

3-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-

6-phenyl-1,3-diazaspiro[4.5]decan-2,4-dione (16)

The free base was obtained in 54% yield aswhite powder; m.p. 149ñ150OC; 1H NMR (300 Hz,CDCl3 δ, ppm): 7.12ñ7.27 (m, 7H), 6.74ñ6.86 (m,3H), 3.19ñ3.26 (t, 2H, , J = 7.10 Hz), 3.13ñ3.17 (t,4H, J = 5.00 Hz), 3.08ñ3.09 (m, 1H), 2.42ñ2.45 (t,4H, J = 5.00 Hz), 2.03ñ2.08 (t, 2H, J = 7.20 Hz),1.76ñ1.91 (m, 5H), 1.42ñ1.58 (m, 3H), 1.13ñ1.27 (m,2H). TLC: Rf = 0.66 (S2); HPLC: Rt = 2.46 (99%).Analysis: calcd. for C27H33N4O2Cl ◊ H2O: C 64.98, H7.07, N 11.23%; found: C 65.13, H 7.15, N 11.14%.

Compounds 17-19 were previously described (9).

1-{3-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]

propyl}-2í,3í-dihydro-2H,5H-spiro[imidazoli-

dine-4,1í-indene]-2,5-dione (20)


The free base was obtained in 60% yield aswhite powder; m.p. 140ñ142OC; 19F NMR (300 Hz,CDCl3 δ, ppm): -62.76 (s, 3F); 1H NMR (300 Hz,CDCl3, δ, ppm): 1.82ñ1.91 (q, 2H, , J = 7.15 Hz)2.18ñ2.28 (m, 1H), 2.40ñ2.45 (t, 2H, J = 7.15 Hz),2.56ñ2.59 (t, 4H, J = 5 Hz), 2.65ñ2.74 (m, 1H),2.98ñ3.10 (m, 1H), 3.16ñ3.20 (t, 4H, J = 5 Hz),3.21ñ3.29 (m, 1H), 3.58ñ3.63 (t, 2H, J = 7.15 Hz),6.50 (s, 1H), 7.02ñ7.11 (m, 3H), 7.17ñ7.35 (m, 5H).TLC: Rf = 0.78 (S2); HPLC: Rt = 2.41 (99%);LC/MS (m/z) 473.5 [M + H]+.

1-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)propyl]-

2í,3í-dihydro-2H,5H-spiro[imidazolidine-4,1í-

indene]-2,5-dione (21)

The free base was obtained in 57% yield ascreamy powder; m.p. 139ñ140OC; H NMR (300 Hz,CDCl3 δ, ppm): 1.82ñ1.91 (q, 2H, J = 7.15 Hz)2.18ñ2.31 (m, 1H), 2.65ñ2.77 (t, 2H, J = 7.00 Hz),2.99ñ3.08 (m, 4H), 3.17ñ3.34 (m, 3H), 3.66ñ3.70 (t,4H, J = 6.80 Hz), 5.89 (s, 1H), 7.04ñ7.34 (m, 8H).TLC: Rf = 0.74 (S2); HPLC: Rt = 1.93 (99%).Analysis: calcd. for C23H25N3O2: C 73.57, H 6.71, N11.19%; found: C 73.23, H 7.06, N 11.07%.

Compounds 22-24 were previously described (9).

1-{3-[4-(3-Trifluoromethylphenyl)piperazin-1-

yl]propyl}-3í,4í-dihydro-2H,2íH,5H-spiro[imida-

zolidine-4,1í-naphthalene]-2,5-dione (25)

The free base was obtained in 56% yield aswhite powder; m.p. 151ñ153OC; 19F NMR (300 Hz,CDCl3 δ, ppm): -62.74 (s, 3F); 1H NMR (300 Hz,CDCl3, δ, ppm): 7.04ñ7.36 (m, 8H), 5.78 (s, 1H),3.40ñ3.69 (t, 2H, J = 7.15 Hz), 3.21ñ3.24 (t, 4H, J =5 Hz), 2.84ñ2.90 (m, 2H), 2.59ñ2.62 (t, 4H, J = 5Hz), 2.45ñ2.49 (t, 2H, J = 7.15 Hz), 2.23ñ2.31 (m,2H), 1.74ñ2.03 (m, 4H). TLC: Rf = 0.81 (S2); HPLC:Rt = 2.48 (99%); LC/MS (m/z) 487.4 [M + H]+.

1-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)propyl]-

3í,4í-dihydro-2H,2íH,5H-spiro[imidazolidine-

4,1í-naphthalene]-2,5-dione (26)

The free base was obtained in 54% yield aswhite powder; m.p. 148ñ151OC; H NMR (300 Hz,CDCl3 δ, ppm): 7.02ñ7.24 (m, 8H), 5.92 (s, 1H),3.67ñ3.73 (t, 4H, J = 7.00 Hz), 2.91ñ3.01 (m, 4H),2.82ñ2.86 (t, 4H, J = 6.90 Hz), 2.22ñ2.31 (m, 2H),1.74ñ1.99 (m, 4H). TLC: Rf = 0.68 (S2); HPLC: Rt =

Table 1. Binding affinity of investigated compounds for serotonin 5-HT1A, 5-HT2A, 5-HT7 and dopaminergic D2 receptors.

Compd.Ki ± SEM [nM]

5-HT1A 5-HT2A 5-HT7 D2

9 1668 ± 75 20 ± 2 NT >10000

10 132 ± 9 147 ± 12 NT NT

11 5211 ± 268 56 ± 7 NT >10000

12 2280 ± 88 462 ± 34 NT NT

13 >10000 >10000 775 ± 16 NT

14 743 ± 56 320 ± 34 NT NT

15 128 ± 15 570 ± 45 NT NT

16 149 ± 9 284 ± 15 249 ± 12 NT

17 98 ± 16* 30 ± 2* NT >10000

18 49 ± 2* 653 ± 100* NT NT

19 38 ± 1* 53 ± 6* 77 ± 8 >10000

20 24 ± 2 58 ± 7 83 ± 5 NT

21 >10000 >10000 84 ± 7 NT

22 88 ± 21* 25 ± 5* NT 675 ± 82

23 23 ± 5* 284 ± 9* NT 965 ± 6

24 350 ± 123* 35 ± 6* 145 ± 15 1800 ± 300

25 22 ± 2 49 ± 4 146 ± 11 >10000

26 768 ± 54 919 ± 46 172 ± 13 NT

* data taken from (9), NT ñ not tested


2.02 (99%). Analysis: calcd. for C24H27N3O2 ◊ H2O:C 70.74, H 7.17, N 10.31%; found: C 70.54, H 6.97,N 10.25%.

Pharmacology in vitroRadioligand binding studies with native 5-

HT1A, 5-HT2A, 5-HT7 and D2 receptors (Table 1)were conducted according to the methods previous-ly described (5). Briefly: 5-HT1A assays used rat hip-pocampal membranes, [3H]-8-OH-DPAT (170Ci/mmol, NEN Chemicals) and 5-HT for non-spe-cific binding; 5-HT2A assays used rat cortical mem-branes, [3H]-ketanserin (88.0 Ci/mmol, NENChemicals) and methysergide for nonspecific bind-ing; 5-HT7 receptor assay was performed using rathypothalamic membranes, [3H]-5-CT (34.5 Ci/mmol; NEN) and 5-HT for non-specific binding andD2 assays used rat striatial membranes, [3H]-spiper-one (15.70 Ci/mmol, NEN Chemicals) and buta-clamol for nonspecific binding. Each compound wastested in triplicate at 7ñ8 concentrations (10ñ11ñ10ñ4

M). The radioactivity was measured by liquid scin-tillation counting (Beckman LS 6500 apparatus) in 4mL scintillation fluid (Akwascynt, BioCare).Binding isotherms were analyzed by nonlinearregression (Prism, GraphPad Software Inc., SanDiego, USA), using the Cheng-Prusoff equation tocalculate Ki values. Results were expressed asmeans of at least two separate experiments.

The extended in vitro evaluation of selectedcompounds (Table 2) was based on the standard

screening procedure (14ñ19). Detailed conditions ofthe assays for respective receptors are shown inTable 3. Briefly, the investigated compounds weretested in screening assay at two final concentrationsof 1.0 and 0.1 µM. The analyzed sample consisted of50 µL of working solution of the tested compound,50 µL of radioligand and 150 µL of a diluted recep-tor source and were transferred to a 96-wellmicroplate. The microplate was covered with a seal-ing tape, mixed and incubated. Reaction mixtureswere filtered on UniFilter 96 GF/C plate and rapid-ly washed with 200 µL of chilled 50 mM Tris-HClbuffer (pH 7.0) using vacuum manifold and 96-wellpipettor. The filtered plate was dried and 30 µL liq-uid scintillator Betaplate Scint was added to eachwell. The radioactivity was measured by MicroBetaTriLux 1450 scintillation counter (PerkinElmer).Results were expressed as percent inhibition of spe-cific binding.

The functional profiles with respect to 5-HT1A

and 5-HT7 receptors were determined at Cerep (LeBois líEveque, 86600 Celle LíEvescault, France)(20). Further methodological details of these studiesare available on the companyís web site (www.cerep.fr).

Moreover, the pharmacological studies tow-ards 5-HT2A were carried out on male Wistar rats((KRF.(WI).WU), Animal House, Faculty ofPharmacy, Jagiellonian University Medical College,KrakÛw) weighing 170ñ350 g. Treatment of labora-tory animals in the present study was in full accor-

Table 2. The extended in vitro pharmacological results of selected compounds for 5-HT6, 5-HT7, D2, D3, α1 receptors and for serotonintransporter (SERT).

% of total binding

Compd. 5-HT6a 5-HT7

b D2c D3

d αα1e SERTf

10-6 10-7 10-6 10-7 10-6 10-7 10-6 10-7 10-6 10-7 10-6 10-7

11 79 37 87 25 13 22 72 21 70 5 14 10

12 63 0 70 15 26 7 89 28 15 14 8 0

13 31 0 51 31 17 15 64 35 21 3 0 0

16 3 0 85 63 17 8 39 4 94 39 7 8

19 7 0 98 88 15 21 22 11 87 60 0 0

20 0 0 94 84 12 23 64 34 72 33 0 19

21 13 0 89 79 0 0 32 22 48 36 11 14

24 33 0 91 76 29 19 38 19 90 64 3 2

25 27 0 93 78 32 19 59 27 69 28 0 5

26 26 0 83 63 4 11 15 15 55 34 13 5

% inhibition of specific binding of reference drugs (10-6/10-7): a Methiothepin (99/99), Serotonin (75/44); b Methiothepin (99/97),Olanzapine (73/54); c Haloperidol (97/99), Olanzapine (92/64); d Haloperidol (98/99), Olanzapine (90/76); e Phentolamine (100/99),Amitriptyline (97/82); f Fluoxetine (96/96), Imipramine (96/91)


dance with the respective Polish regulations.All procedures were conducted according toguidelines of ICLAS (International Council onLaboratory Animal Science) and approved bythe Local Ethics Committee on AnimalExperimentation.

Molecular modeling

The homology models of human 5-HT1A

and 5-HT7 serotonin receptors used hereinwere generated based on developed and well-validated method and described in previouslypublished papers (10, 21, 22). Glide, inducedfit docking, LigPrep and Protein PreparationWizard were implemented in Schro®dingerSuite software, which was licensed forJagiellonian University Collegium Medicum.


The designed spirohydantoins (9ñ26)were synthesized in a multi-step proceduresummarized in Scheme 1. The core spirohy-dantoins were obtained in a Bucherer-Bergreaction (1ñ4), following the alkylation at posi-tion-N3 of a heterocyclic ring (5ñ8) (9, 11).Then, coupling with differently substitutedphenylpiperazines (9ñ12, 15ñ20, 22ñ25) ortetrahydroisoquinoline (13, 21, 26) gave thefinal compounds 9ñ26 in moderate yields(52ñ72%). All the final products were obtainedas racemic mixtures and for the further phar-macological studies they were transformed intowater-soluble hydrochloride salts.

In accordance with the strategy of themulti-receptor ligands, the affinity for serotoninand dopamine receptors and for serotonin trans-porters was determined (Tables 1, 2).Generally, the comparison of substituent at 5position of the hydantoin moiety showed anoticeable impact on receptor binding proper-ties. The results show that the fusion of an aro-matic area with the cycloalkane ring as a rigidskeleton (17ñ20, 22ñ25) significantly increasedthe binding to serotonin 5-HT1A and 5-HT7

receptor sites (Table 1), whereas the introduc-tion of the phenyl ring to 5-cyclohexane-spiro-hydantoin as a flexible fragment (9ñ16) result-ed in a decreased affinity for those receptors.

Moreover, it seems that also the secondaryamine and the nature of the substituents inphenyl ring had a crucial impact on the affinityto the receptors of the tested compounds. Theresults presented in Table 1 revealed that

Tab

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almost all selected spirohydantoin derivatives with atetralin or indane moiety in the amide part (19ñ21,24ñ26) showed moderate activity toward 5-HT7

receptors. It seems that this activity toward 5-HT7

receptors is the result of the occurrence in the struc-ture of both the amide mentioned above and an elec-tron withdrawing group/atom in the phenylpiper-azine moiety or tetrahydroisoquinoline fragment.Moreover, some compounds with an unsubstitutedphenylpiperazine fragment (9, 17, 22) or their coun-terparts with 3-chloro (11, 19, 24) or 3-trifluromethylsubstituents (20, 25) possessed a moderate-to-highaffinity for 5-HT2A; two of these show high selectiv-ity for 5-HT2A receptors (9 and 11). Furthermore, thepresence of an indane and tetralin moiety and aphenylpiperazine fragment in the structure of theinvestigated compounds had a positive impact on thebinding at 5-HT1A receptor sites (17ñ20, 22, 23, 25).The tetrahydroisoquinoline derivatives (13, 21, 26)showed no affinity for 5-HT1A/2A receptors, but amoderate affinity for 5-HT7 receptors.

Additionally, the spirohydantoin derivativeswith a withdrawing group (Cl, CF3) substituted intoa phenylpiperazine moiety or with a tetrahydroiso-quinoline fragment (11ñ13, 16, 19ñ21, 24ñ25) werechosen for screening evaluation and their affinity fordopamine (D2, D3) and adrenergic (α1) receptors andserotonin transporters (SERT) was determined(Table 2). In this screening study, all the selectedcompounds showed a high-to-moderate affinity for5-HT7 receptors. However, apart from compound 11

which had weak affinity for 5-HT6 receptors, theother derivatives were inactive. The tested com-pounds exhibited a low or no affinity for dopamineD2 receptors and were practically devoid of anyaffinity for SERT. Moreover, compounds contain-ing a 3-trifluromethylphenylpiperazine fragment(12, 20 and 25) or a 8-phenyl-cyclohexyl moiety

(11ñ13) demonstrated a moderate affinity fordopamine D3 receptors, unlike the other spirohydan-toin derivatives. In this screening test, most of thepotent serotonin receptor ligands revealed a high-to-moderate affinity for adrenergic α1 receptors.

For further functional and molecular modelingstudies, one (19) of the two counterparts (19, 20)which exhibited a sustainable affinity for serotonin5-HT1A/2A/7 receptors and a moderate affinity fordopaminergic D3 receptors was chosen as an exam-ple. Moreover, for functional profile evaluation, thetwo compounds (9, 11) with the highest selectivityand affinity for 5-HT2A receptors were selected.

The binding mode of the lead compound 19 atthe sites of serotonin 5-HT1A and 5-HT7 receptorswas analyzed in detail, as a representative one. Tothis end, the previously developed homology mod-els of the receptors were used (20). The tested com-pound was synthesized in a racemic form; neverthe-less, predominantly better scores and more favor-able interactions in both targets were observed forthe S enantiomer, and therefore its binding modewas described. The binding mode of the ligand inthe two receptors was shown to be consistent bothwith the common one for monoaminergic receptorligands and with previous results (20, 23). The com-pound 19 molecule in the 5-HT1A receptor adoptedlinear conformation, extending from the deeper cav-ity formed by transmembrane helices (TMHs) 3ñ6to the second interaction pocket located betweenTMHs 1, 2 and 7. In the 5-HT7 receptor, the mole-cule bent to find interactions in less spatial pocketsituated closer to TMH3. The main anchoring inter-action in both sites was a charge-reinforced hydro-gen bond between the protonated nitrogen atom ofthe ligand and the carboxyl group of Asp3.32, aswell as CH-π interactions of the arylpiperazine andaromatic amino acid cluster of the deeper cavity,

Scheme 1. Synthetic pathways of compounds 9ñ26. Reagents and conditions: (i) KCN, (NH4)2CO3, 50% ethyl alcohol, 28 h, 56OC; (ii)K2CO3, KJ, acetone, reflux 20 h (iii), -substituted piperazine derivatives or tetrahydroisoquinoline, 96% ethyl or butyl (compd. 13) alco-hol or 2-methoxyethanol (compd. 16), reflux 40 h


mainly Phe6.52 (Fig. 1). The spirohydantoin frag-ment of the molecule occupied the additional cavityand found both the hydrophobic and polar, favorablecontacts there, which varied depending on the recep-tor type. For 5-HT1A receptors, the carbonyl oxygenof hydantoin formed an h-bond with the NH2 groupof Asn7.39, while the aromatic ring of indane inter-acted with the phenyl ring of Tyr2.64 (π-π stacking,Fig. 1A). In the 5-HT7 receptor, the latter fragmentformed an analogous interaction with Phe3.28,although the conformation seems to be suboptimal,since the complex lacks additional favorable inter-actions of h-bond nature (e.g., with Arg7.36), whichmay contribute to the relatively lower affinity ofcompound 19 for this site (Fig. 1B). On the otherhand, the m-Cl substituent at the phenylpiperazinefragment is devoid of polar interactions with, forexample, Ser5.42 or Lys191 from the second extra-cellular loop (ECL), which, if present, might haveincreased affinity for 5-HT1A receptors.

On the basis of binding affinity results, com-pound 19 was selected as an example for functional

in vitro screening toward serotonin 5-HT1A and 5-HT7 receptors. Compound 19 was classified (Fig. 2)as an agonist of 5-HT1A receptors (59.5% in 1.0E-06M) and a weak antagonist of 5-HT7 receptors(41.1% in 1.0E -06 M).

The antagonist activity of compounds 9 and 11

toward 5-HT2A-receptors present in rat aorta wasassessed via the inhibition of serotonin-induced con-tractions (Fig. 3). Both compounds 9 and 11 dis-played an ability to block the contractions induced byserotonin, giving a pKB value estimate of 7.665 ±0.034 and 7.110 ± 0.048, respectively. It is noticeablethat the affinity from the functional tests for the stud-ied compounds was in the same concentration rangeas that determined in the radioligand binding assay.

CONCLUSION

In conclusion, we described the synthesis of8/6-phenyl-1,3-diazaspiro[4.5]decan-2,4-diones and2í,3í-dihydro-2H,5H/3í,4í-dihydro-2H,2íH,5H-spiro[imidazolidine-4,1í-indene/naphthalene]-2,5-

Figure 1. Binding modes of compound 19 in the binding sites of 5-HT1A (A) and 5-HT7 (B) receptors. Amino acid residues engaged in lig-and binding (within 4� from the ligand atoms) are shown as thick sticks. Dotted yellow lines represent H-bonds with polar residues. Forthe sake of clarity a part of ECL2 was hidden. TMH ñ transmembrane helix; ECL ñ extracellular loop


diones connected with an arylpiperazine or tetrahy-droisoquinoline fragment by the propylene carbonchain, which have proven to be potent serotoninreceptor ligands. The obtained pharmacologicalresults demonstrated that the introduction of an aro-matic area into the cycloalkane ring as rigid frag-ment (indane or tetralin) at position 5 of imidazoli-dine-2,4-dione noticeably increases the affinity for

serotonin receptors. Moreover, the presence of awithdrawing group substituted into a phenylpiper-azine moiety had a positive impact on the binding at5-HT1A, 5-HT2A, 5-HT7 receptor sites. In contrast,the replacement of arylpiperazine fragment withtetrahydroisoquinoline moiety resulted in decreasedaffinity for 5-HT1A/2A receptors. Therefore, based onpreliminary pharmacological research, two com-

Figure 2. Functional properties for compound 19, percent of control agonist response (on the top) and percent inhibition of control agonistresponse (on the bottom)

Figure 3. Concentration-response curves to serotonin in the rat aorta in the absence (■) or presence of compound 9 (on the left, ▼100 nM);and 11 (on the right, ▼100 nM); results are expressed as percentage of the maximal response to KCl depolarizing solution. Each point rep-resents the mean ± SEM (n = 4)


pounds (19, 20) which possessed high affinity forserotonin 5-HT1A, 5-HT2A, 5-HT7 receptors andmoderate affinity for dopaminergic D3 receptorswere selected for further pharmacological studies.Furthermore, interactions with serotonin 5-HT1A/7

were described for compound 19, which behaved asa 5-HT1A agonist and weak 5-HT7 antagonist.Additionally, from among the compounds withmulti-receptor profile, we obtained two compounds(9 and 11) with suboptimal affinity which behave asantagonists of 5-HT2A receptors.

Acknowledgments

This study was financially supported byNational Science Center grants (No. DECñ2011/01/B/NZ4/00695, DECñ2012/07/B/NZ7/01173),and by Funds for Statutory Activity of JagiellonianUniversity Medical College (No. K/ZDS/004652).

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Received: 5. 01. 2016


In the view of global health concerns over drugresistant microorganisms and life threatening dis-eases, the need for new and effective bioactive com-pounds is ever increasing. Since ages, man hasrelied on sources like plants and microbes for drugdiscovery. Among microorganisms, fungi haveserved as fountainhead of bioactive metabolites (1).In this regard, endophytic fungi have received con-siderable attention in recent years.

Endophytes are the microorganisms that inhab-it living tissues of higher plants without producingany disease symptoms (2). The mutualistic relation-ship between endophytes and their host plantenables the endophytes to produce diverse classes ofsecondary metabolites like alkaloids, terpenoids,steroids, etc. (3-5). Once isolated and characterized,these compounds can be employed as agrochemi-

cals, antibiotics, anticancer or antioxidant agents(6). Literature survey suggests relatively high per-centage of novel chemical entity production byendophytes (51%) as compared to soil fungus (38%)(7).

Studies on the bioactive potential of endo-phytes are still in their stage of infancy (8). At pres-ent, these investigations have been intensified, withspecial focus on endophytes of medicinal plants.Northern areas of Pakistan have huge biodiversity ofplants with significant pharmacological importance(9). This prompted us to study the bioactive poten-tial of the endophytes of Taxus fauna of the region.Therefore, present study was aimed to investigatethe production and potential of antimicrobial pep-tides from selected endophytic fungus under differ-ent experimental conditions.

NATURAL DRUGS

PRODUCTION OF ANTIMICROBIAL PEPTIDES BY EPICOCCUM SP. NFW1:AN ENDOPHYTE OF TAXUS FAUNA

MUNIBA JADOON1*, NIGHAT FATIMA1,2,3, SIDRA MURTAZA1, LENG CHEE CHANG4, NAEEM ALI1 and SAFIA AHMED1

1Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, 45320, Islamabad, Pakistan

2Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 3Department of Pharmacy, COMSATS Institute of Information Technology (CIIT),

Manshehra Road, Abbottabad, Pakistan (Present address)4Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy,

University of Hawaii Hilo, Hilo, HI, 96720, United States

Abstract: An endophytic fungus NFW1, possessing antimicrobial activity against bacterial and fungalpathogens, was isolated from indigenous Taxus fauna. Phylogenetic analysis coupled with cultural and mor-phological characteristics revealed that endophyte NFW1 closely resembles Epicoccum sp. It showed optimumgrowth and antimicrobial activity in mineral salt medium TM, incubation temperature 25OC, incubation time 15days and pH 6.5. Antimicrobial peptides were precipitated with 80% ammonium sulfate and expressed signif-icant inhibitory effect against Staphylococcus aureus (ATCC6538) and Candida albicans (CI.I 4043). It alsoinhibited growth of Streptomyces 85E in hyphae formation inhibition assay showing potential as protein kinaseinhibitor. Gel filtration chromatography on Sephadex G-75, followed by sodium dodecyl sulfate polyacry-lamide gel electrophoresis resolved the crude precipitate into three fractions of molecular mass 32 kDa, 44 kDaand 70 kDa. The study concludes that endophytic fungi associated with indigenous Taxus species possess prom-ising antimicrobial activities and should be exploited as source of novel antimicrobial agents.

Keywords: antimicrobial, endophyte, Epicoccum sp., peptide, Taxus fauna

1555

* Corresponding author: e-mail: [email protected]; phone: +92-51-90643009

1556 MUNIBA JADOON et al.

EXPERIMENTAL

Materials and Methods

A total of 16 strains of endophytic fungi, iso-lated previously from Taxus fauna of Himalayanregion of Pakistan were obtained from MicrobiologyResearch Lab (MRL) of Quaid-i-Azam University,Islamabad, Pakistan. Cultures were maintained onpotato dextrose agar (PDA) at 4OC.

Culture activation and initial screening

All the endophytic fungal strains were re-culti-vated on PDA for 5-7 days at 25OC. From the freshcultures, three pieces (0.5 ◊ 0.5 cm2) of mycelialagar plugs were inoculated in 200 mL of Sabourauddextrose broth (SDB). Flasks were run on a shakerincubator at 150 rpm and 25OC for 14 days. Afterincubation, broth culture was filtered and filtratewas screened for antimicrobial activity against apanel of microbial strains.

Antimicrobial activity

Filtrate was evaluated for activity against twoGram positive bacteria- Staphylococcus aureus(ATCC 6538), Micrococcus luteus (ATCC 10240), 3

Gram negative bacteria Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC 9721),Klebsiella pneumoniae (ATCC 13883) and a fungalhuman pathogen Candida albicans (CI.I 4043).Fungal plant pathogens were Aspergillus flavus(FCBP 0064), Aspergillus fumigatus (FCBP 66),Aspergillus niger (FCBP 0198), Aspergillus terreus(FJ654431). All the cultures were obtained fromMRL, Quaid-i-Azam University, Islamabad.

Agar well diffusion assay

Antimicrobial activity of the fungal cultureswas determined by conventional agar well diffusionassay (10). Pathogenic fungal and bacterial suspen-sions were prepared and adjusted to inoculum size106 colony forming units (CFU/mL). Microbial sus-pensions were spread over plates containing nutrientagar (NA) and PDA and wells of 8 mm diameterwere made. Equal volume of filtrate (100 µL/well)was loaded into each well and clear zone of inhibi-tion were measured after 24-72 h. Tetracycline (30µg/well; Sigma Aldrich: percentage purity = 98.0%)and nystatin (100 µg/well; Sigma Aldrich: contains4,400 USP units/mg) were used as positive controlfor antibacterial and antifungal activity, respectively.

Table 1. Antimicrobial activity of endophytic fungi isolated from Taxus fauna against bacterial and fungal pathogens

Diameter of zone of inhibition (mm)

Endophyte E. K. M. P. S. C. A. A. A. A.coli pneumonia luteus aeruginosa aureus albicans flavus fumigatus niger terreus

NFW1 12.8 ± 10.2 10.3 ± 0.4 11.8 ± 0.5 +2 13.2 ± 0.2 12.9 ± 0.5 9.8 ± 0.5 10.2 ± 0.2 + 11.2 ± 0.4

NFW2 -3 - - - - - - - - -

NFW3 12.2 ± 0.3 9.8 ± 1.0 11.6 ± 0.2 - 12.7 ± 0.6 10.5 ± 0.3 9.8 ± 1.6 10.1 ± 0.4 8.8 ± 0.2 10.4 ± 0.6

NFW4 10.5 ± 0.8 - 8.9 ± 0.3 - - 9.9 ± 0.2 - - - -

NFW5 - - - - - - - - - -

NFW6 9.9 ± 0.5 9.6 ± 0.6 10.2 ± 0.8 - 10.3 ± 0.7 9.5 ± 0.3 9.2 ± 0.6 9.8 ± 0.2 - -

NFW7 11.2 ± 0.7 9.8 ± 1.3 11.2 ± 0.1 - 11.7 ± 0.9 10.8 ± 0.7 9.1 ± 1.0 9.4 ± 0.6 - -

NFW8 10.5 ± 0.4 + + - 8.8 ± 0.3 - - - - -

NFW9 + - 9.1 ± 0.8 - - 8.9 ± 0.8 + + - -

NFW11 - - - - - - - - - -

NFW12 - - + - + - 9.2 ± 0.5 8.8 ± 0.1 - -

NFL1 8.9 ± 1.1 - - - - - - - - -

NFL2 - - - - - + + - - -

NFL3 - - - - - - - - - -

NFL5 - - - - - - - - - -

NFL6 9.5 ± 0.6 9.1 ± 0.4 - - - + - - - -

Control4 21.4 ± 0.6 20.6 ± 0.5 23.1 ± 0.8 15.6 ± 0.4 24.1 ± 0.7 22.8 ± 0.9 21.3 ± 0.4 26.9 ± 0.6 21.6 ± 0.813.2 ± 0.6

1 Standard deviation. 2 Zone of inhibition less than 8.5 mm. 3 No inhibition. 4 tetracycline (30 µg/well) and nystatin (100 µg/well) was usedas positive control against bacterial and fungal pathogens, respectively. Blank media (100 µL/well) served as negative control.

Production of antimicrobial peptides by Epicoccum sp. NFW1... 1557

Based on initial screening, fungal endophyteNFW1 was selected for further studies.

Identification of the endophyte

Endophytic fungus NFW1 was identified on thebasis of morphological features (microscopic/ macro-scopic) and internal transcribed spacer (ITS) region

sequencing of ribosomal DNA (rDNA). For taxonom-ic identification, DNA was extracted by CTAB extrac-tion method (11). A pair of universal primers ITS1(sequence) and ITS4 was used to amplify the targetgenes. PCR was carried out in a programmable MJMiniTM gradient thermal cycler (Bio-Rad LaboratoriesInc., USA). For PCR mixture (25 µL), 5 µL of DNA

Figure 1. Phylogenetic relationship between Epicoccum sp. NFW1 and closely related fungal strains based on ITS-rDNA sequences. Theevolutionary history was inferred using Neighbour-Joining (NJ). Number above or below the branches indicate bootstrap values of NJ from1000 bootstrap replicates

Table 2. Antimicrobial activity of protein precipitate of Epicoccum sp. NFW1 against bacterial and fungalpathogens.

Diameter of zone of inhibition (mm)Test organisms

Crude protein Tetracycline1 Nystatin(4 mg/well) (30 µg/well) (100 µg/well)

E. coli 15.2 ± 20.3 21.4 ± 0.6 x3

K. pneumonia 9.8 ± 0.6 21.6 ± 0.8 x

M. luteus -4 15.6 ± 0.3 x

P. aeruginosa - 22.8 ± 0.8 x

S. aureus 20.8 ± 0.4 24.1 ± 0.7 x

C. albicans 18.1 ± 0.7 x 22.8 ± 0.1

A. flavus - x 21.2 ± 0.8

A. fumigatus 14.3 ± 0.8 x 27.3 ± 1.0

A. niger - x 24.8 ± 0.2

A. terreus - x 21.6 ± 0.8

1Tetracycline and nystatin were used as positive control in antibacterial and antifungal assay respectively.Phosphate buffer (0.1 M, pH 6.5) served as negative control. 2Standard deviation (SD). 3Not tested. 4No inhibi-tion.


Figure 2a. Effect of media on growth and antimicrobial activity of Epicoccum sp. NFW1

Figure 2b. Effect of incubation time on growth and antimicrobial activity of Epicoccum sp. NFW1

Figure 2c. Effect of temperature (∞C) on growth and antimicrobial activity of Epicoccum sp. NFW1

template was mixed with 3 µL of 25 mM MgCl2, 100µM of each dNTP, 25 pM of each primer and 1 U ofTaq DNA polymerase. All the chemicals/primerswere purchased from Fermentas (Fermentas, ThermoScientific, USA). The amplification was carried out at95OC for 60 s followed by 35 cycles at 95OC for 30 s,55OC for 60 s and 72OC for 60 s, with a final cycle at

72OC for 6 min (12). Purified products were sequencedusing Big Dye terminator cycle sequencing kit v.3.1(Applied BioSystems, USA) and resolved on anApplied Biosystems model 3730XL automated DNAsequencing system (Applied Biosystems, USA) atMacrogen, Korea. NCBI BLAST was used to searchsimilar sequences in GenBank and identified sequence


was submitted at NCBI GenBank to obtain the acces-sion number. Phylogenetic tree was constructed usingsoftware MEGA version 6 based on Neighbor-Joining(NJ) method (13).

Optimization of process parameters

Standardization of media

Standardization of media for optimum growthand antimicrobial activity was carried out usingthree different media.

MID (g/L): sucrose 3 g, yeast extract 0.25 g,ammonium tartarate 5 g, calcium nitrate tetrahydrate0.5 g, potassium nitrate 80 mg, magnesium sulfateheptahydrate 360 mg, potassium chloride 60 mg,sodium dihydrogen phosphate 20 mg, ferric chloride2 mg, magnesium sulfate 5 mg, zinc sulfate hep-tahydrate 3 mg, hydroborate 1.4 mg, potassiumiodide 0.7 mg (14).

TM (g/L): sucrose, 40; phenylalanine, 0.01;peptone, 0.5; yeast extract, 0.8; (NH4)2SO4, 3;MgSO4◊7H2O, 0.5; KH2PO4, 2; NaCl, 0.6;CH3COONa, 0.5; C6H5COONa, 0.1; (15).

Sabouraud dextrose broth (Oxoid, UK g/L):dextrose 20, pancreatic digest of casein 5, pepticdigest of animal tissue 5, (pH 5.6 ± 2).

The biomass accumulation and antimicrobialactivity was determined after 14 days of incubationat 25OC and 150 rpm. Biomass accumulation wasdetermined by drying the mycelial mass at 50OCuntil constant weight was obtained and expressed asg/L. Antimicrobial activity was expressed as diame-ter of zone of inhibition, measured in millimetres(mm).

Effect of incubation time

Strain NFW1 was inoculated into selectedmedia and run on a shaker incubator at 25OC and 150

rpm. Optimum incubation time for growth andactivity was determined by comparing the dryweight and zone of inhibition of the filtered brothevery 72 h for 24 days.

Effect of incubation temperature

The optimum temperature for cell growth andantimicrobial activity was determined by comparingbiomass and antimicrobial activity at temperaturerange 10-40OC. All other conditions were similar.

Effect of pH

Initial pH of the growth media was adjustedfrom 5 to 8 and flasks were incubated keeping all theother conditions at optimum level. Optimum pH wasdetermined by comparing dry weight and diameterof zone of inhibition as described earlier.

Protein precipitation

The fungal strain was grown in mineral saltmedia TM under optimized growth conditions.Protein precipitation was carried out using ammoni-um sulfate as reported by Nadhman et al. (16). Cellfree supernatant was centrifuged at 10,000 ◊ g for 20min before ammonium sulfate was added in increas-ing concentration (10-80%). Pellet was collected bycentrifugation at 10,000 ◊ g for 15 min and dis-solved in minimum amount of 0.1 M phosphatebuffer, pH 6.5. Protein estimation was conducted asdescribed by Lowry et al. (17).

Biological evaluation of crude protein precipitate


Antimicrobial activity of the crude protein pre-cipitate was determined by agar well diffusion assayas described earlier (10). Assay was performed intriplicate and the mean of the values noted.

Figure 2d. Effect of pH on growth and antimicrobial activity of Epicoccum sp. NFW1


HFI inhibition activity

Hyphae formation inhibition (HFI) assay wasperformed as reported by Waters et al. (18).Mycelial fragments of Streptomyces 85E werespread on ISP4 (International Streptomyces Project)agar media to produce bacterial lawn. Paper disk (7mm) impregnated with 80 mg of the test sample (invarying concentrations) were placed on the plate.Zone of inhibition around each disc was noted after30 h of incubation at 30OC. Two types of phenotypescould be observed: a clear zone of inhibition and/ora bald phenotype around the disc. Surfactin (20mg/disk-SIGMA) was used as positive control while0.1 M phosphate buffer (pH 6.5) served as negativecontrol. Concentration with zone of inhibitiongreater than 8 mm was considered positive. Assaywas run in triplicate.

Antioxidant activity

Antioxidant activity of the precipitate wasdetermined by using diphenylpicrylhydrazyl (DPPHassay) as described by Kanwal et al. (19). Briefly,2800 µL of 0.1 mM DPPH solution (in 82%methanol) was mixed with 200 µL of test sample (indistilled water). Solution was serially diluted toobtain final concentration of 100, 50, 25, 5, 2 and 1ppm. Vials were incubated in dark at 37OC for 1 h.After incubation, absorbance of the mixture wasmeasured at 517 nm on UV/Visible spectropho-tometer (DAD Agilent 8453). A mixture of 2800 µL

of 82% methanol and 200 µL of distilled waterserved as blank while ascorbic acid was used as pos-itive control. The percentage scavenging of DPPHby protein precipitate, run as three independentexperiments, was calculated using the following for-mula:

% Scavenging = [(A - B)/A] ◊ 100where A = absorbance of negative control, B =absorbance of test sample

Gel permeation chromatography (Sephadex G-

75)

Gel permeation chromatography was per-formed as reported by Nadhman et al. (16). Crudeprotein precipitate was loaded on Sephadex G-75column and eluted by using phosphate buffer (0.1M, pH 6.5). Fractions showing antimicrobial activi-ty were pooled, lyophilized and subjected to SDS-PAGE analysis.

Sodium dodecyl sulfate - polyacryl amide gel

electrophoresis (SDS-PAGE)

SDS-PAGE was performed using 12% w/vseparating gel (polyacryl amide) and 5% w/v stack-ing gel as proposed by Laemmli et al. (20). Sampleswere run along with the standard protein marker of10-170 kDa (Fermentas, USA). Gel was stainedwith Coomassie brilliant blue R-250 (Sigma-Aldrich) and visualized on gel documentation sys-tem (Bio-Rad, USA).

RESULTS

Initial screening showed that 11 of 16 isolateswere active against one or more test microbial strain(Table 1). The strongest antimicrobial activity wasexpressed by strain NFW1 by forming clear zone of9.8 mm to 13.6 mm. Therefore, this strain was pri-oritized for further studies.

Colonies of NFW1 are initially pink on PDAmedium and slowly turn peach in color with dark pinkand greenish edges. Reverse appears dark peach withslightly wavy margins. Microscopic and macroscopicfeatures of the isolate show strong similarity withEpicoccum sp. A sequence similarity of 89% wasobserved when compared with previously submitted

Table 3. Antioxidant activity of protein precipitate of Epicoccum sp. NFW1.

Test sample%age scavenging at different test concentrations (ppm)

100 50 25 10 5 2 1

Crude protein 55.4 41.3 22.7 0 0 0 0

Ascorbic acid 95.1 94.8 90.0 86.4 44.7 17.6 1.77

Table 4. Inhibitory activity of protein precipitate of Epicoccum sp.NFW1 against Streptomyces 85E (HFI Assay).

Test concentration Zone of(µg/disk) inhibition (mm)

80 25 ± 10.8

40 20 ± 0.2

20 16 ± 0.6

10 11 ± 1.1

5 8 ± 0.5

Surfactin2 30 ± 0.6

1Standard deviation (SD). 2Surfactin (5 µg/disk, positive control)formed a bald zone of 30 mm. Phosphate buffer (0.1 M, pH 6.5)served as negative control.


sequences for Epicoccum sp. in NCBI GenBank.Therefore; fungal endophyte NFW1 is identified asEpicoccum sp.; sequence submitted in NBCIGenBank under accession number JX402049 (Fig. 1).

In our study, optimization of fermentation con-ditions showed that mineral salt media TM (Fig. 2a),incubation time 15 days (Fig. 2b), temperature 25OC(Fig. 2c) and pH 6.5 (Fig. 2d) is most suited forgrowth and production of antimicrobial substancesby NFW1.

Epicoccum sp. NFW1 showed protein precipi-tation at 80% ammonium sulfate saturation. Crudeprecipitate expressed significant antimicrobial activ-ity against different pathogens (Table 2). Maximuminhibitory effect was observed againstStaphylococcus aureus ATCC6538 (20.8 mm) fol-lowed by that against Candida albicans CI.I 4043(18.1 mm). Antioxidant activity of crude precipitatewas negligible at the concentrations tested (Table 3).In hypae formation inhibition assay, a clear zone of8-25 mm inhibition was observed at concentration 5µg/disk ñ 80 mg/disk (Table 4). No activity wasobserved below 5 µg/disk.

Gel permeation chromatography resolved thecrude precipitate into 3 fractions active only against

Staphylococcus aureus ATCC6538 and Candidaalbicans CI.I 4043. Antimicrobial activity against S.aureus ranged from 10 to 23 mm while that againstC. albicans was 8.9 to 11.5 mm. On SDS-PAGE,fractions resolved into single band of molecularweight 32, 43 and 70 kDa, respectively (Fig. 3).

DISCUSSION

Endophytic fungi are a novel group of microor-ganisms possessing promising biological potential.Despite continuous studies on isolation and charac-terization of these organisms, their bioactive poten-tial still remains largely untapped (8). In this study,an attempt was made to investigate the bioactivepotential of protein precipitate of endophytic fungi.Since their initial discovery, many studies reportedthe antimicrobial potential of endophytic fungi (8,21, 22). Initial screening of 15 endophytic fungishowed that these organisms could be a reliablesource of antibacterial substances. Our findings aresupported by studies from neighboring country -India, which describe interesting bioactive potentialof endophytes isolated from native Taxus species (8,23-26).

Most active endophyte, NFW1 is identified asEpicoccum sp. Epicoccum sp. is reported endophyteof various plants like Taxus baccata, Taxus brevifo-lia, Pinus wallichiana, apple, Vitis vinifera, orchi-dacease sp. and sugar cane (14, 27-30).

In any fermentation process, physiochemicalparameters like culture media, incubation time, tem-perature, pH, etc. are always crucial for significantresults. Standardization of media showed that min-eral salt media TM under initial pH 6.5; is most suit-ed for growth and antimicrobial activity of NFW1.Studies on media optimization have shown that car-bon, nitrogen source and amino acids particularlyaffect growth and antimicrobial activity of cultivat-ed fungi (31, 32). Previous studies report yeastextract amended media and pH 6 to neutral as favor-able for maximum production of antimicrobialagents (21, 32, 33).

Epicoccum sp. NFW1 expressed optimumgrowth and antimicrobial activity when incubatedfor 15 days, at temperature 25ñ30OC. Studies showthat incubation time of 10-12 days and temperaturerange 25ñ30OC as optimum for growth and antimi-crobial activity of endophyte (24, 32). Stinson et al.(34) reported similar results for endophyticGliocladium sp. Maximum antimicrobial activity isusually found as fungi reach stationary phase (32).This is because synthesis of secondary metabolitesis accelerated as nutritional factors diminish thereby

Figure 3. SDS-PAGE (12% gel) of the antimicrobial proteinsobtained from Epicoccum sp. NFW1. Lane 1 - Fraction 1 (32kDa), Lane 2 - Fraction 2 (44 k Da), Lane 3 - Fraction 3 (70 kDa),Lane 4 - Molecular mass standard (Ladder: 10 kDa - 70 kDa). Gelwas stained with Coomassie brilliant blue R-250


limiting growth and promoting biological activity.Similarly, temperature lower than optimum ceasesthe metabolic activity while higher kills the fungalcells (32).

Crude protein precipitate, obtained under 80%ammonium sulfate concentration, expressed broadspectrum antimicrobial activity. Epicoccum sp. hasprovided important antimicrobials like epicorazine,epicoccamides and epicoccins (35-39). Our findingsencouraged us to evaluate the potential of NFW1 asprotein kinase inhibitor. An HFI assay was per-formed where a clear zone was observed under dif-ferent concentrations. A clear zone indicates that theagent inhibits growth as well as sporulation, rapidlyindicating the presence of signal transductioninhibitors (18). It is hypothesized that compoundsinhibiting hyphae formation in Streptomyces 85Ecould potentially block cancer proliferation andmycobacterial infections (18). Therefore, proteinprecipitate of Epicoccum sp. NFW1 could be evalu-ated further for its role in targeted drug develop-ment. Aly et al. (40) reported production of proteinkinase inhibitors from fungal endophyte Stemp-hylium botryosum.

Crude precipitate revealed 3 active fractionswhen purified using Spehadex G-75. Each fractionresolved into single band on SDS-PAGE. At pres-ent, we are investigating the amino acid sequenceof the purified components and their status asindividual or part of same protein. Researchershave successfully isolated novel wide spectrumpeptidal antibiotics Mumbicins (1269 Da - 1326Da) and Kakadumycins from endophyticStreptomyces sp. (41, 42). Strobel et al. (43)reported production of antifungal proteinCryptocandin, 1079 Da, from endophytic fungusCryptosporiopsis. Wu et al., (44) isolated singlechain fibrinolytic enzyme of 28 kDa from endo-phytic Fusarium sp. CPCC 480097.

In conclusion, protein precipitate obtainedfrom Epicoccum sp. NFW1 presents significantantimicrobial activity suggesting the promisingpotential of the indigenous endophytes as source ofbioactive peptides. Since this is preliminary investi-gation, further studies on the physiological potentialof these bioactive proteins is imperative along withtheir sequencing, cloning and possible role in target-ed drug development.

Acknowledgments

This work was supported technically andfinancially by Microbiology Research Lab,Department of Microbiology, Quaid-i-Azam

University, Islamabad, Pakistan and College ofPharmacy, University of Hawaii, Hawaii, USA.

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Received: 28. 09. 2015


Water chestnut was first formally described byCarl Linne as the Trapa natans L. (1753). This plantbelongs to the genus Trapa, family TrapaceaeDumortier (Syn.: Hydrocariacea Raimann, orderMyrtales) (1, 2). According to JankoviÊ (2, 3) theterm T. natans L. is the old name for the complexEuropean and East Asian species. In the theory offlora of western and central Balkans, based on theevolution data of genus Trapa fruit, it includes twosections: Longicarpa and Brevicarpa (2). There arenumerous Latin names for species of genus Trapa aswell as multiple traditional names. SectionLongicarpa contains three species with long (tines)prongs: T. annosa, T. longicarpa M. Jank. and T.europaea Fleroff. In Serbia, the section Brevicarpaincludes: T. brevicarpa M. Jank., with short prongs.Based on molecular studies, in the new classifica-tion, the genus Trapa belongs to the family

Lythracaeae, order Myrtales (4). Thus Porcher et al.(5) point out that the general conflict about classifi-cation of water chestnut reflects unreconciled viewsregarding the current systematics of the genus andspecies of water chestnut (Melbourne UniversityProject Multilingual, multiscript Plant Name DataBase) (6).

T. natans is reported throughout most ofEurope, in the north to southern Sweden and in theeast to European Russia. Outside Europe, it occursthroughout most of Asia to China, the Philippinesand Malaysia in the east. Apparently, it was intro-duced into Australia and North America where it iswidespread at present and there is a possibility for itbeing invasive. It is difficult to interpret informationon the distribution of this species with any confi-dence due to taxonomic confusion. Thus, for exam-ple, according to JankoviÊ and BlaûenËiÊ (6), the

ANTIMICROBIAL AND ANTIBIOFILM EFFECTS OF EXTRACTS FROMTRAPA NATANS L., EVALUATION OF TOTAL PHENOLIC AND FLAVONOID

CONTENTS AND GC-MS ANALYSIS

IVANA D. RADOJEVI∆1*, SAVA M. VASI∆1, MILAN S. DEKI∆2,3, NIKO S. RADULOVI∆2,GORICA T. –ELI∆1, JELENA S. –UR–EVI∆1 and LJILJANA R. »OMI∆1

1Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja DomanoviÊa 12, 34000 Kragujevac, Republic of Serbia

2Department of Chemistry, Faculty of Science and Mathematics, University of Niö, Viöegradska 33, 18000 Niö, Republic of Serbia

3Department of Chemical and Technological Sciences, State University of Novi Pazar, Vuka KaradûiÊa bb, 36300 Novi Pazar, Republic of Serbia

Abstract: Research conducted in this study shows the applied in vitro antimicrobial and antibiofilm activity ofthe four extracts isolated from Trapa natans L. leaves. In this study, different methods were used (microdilu-tion, tissue culture plate, different colorimetric methods, GC-FID and GC-MS analysis). While the water extractdidnít show antibacterial activity, the acetone extract showed the strongest one. The same activity in the caseof Pseudomonas aeruginosa (MIC was 313 µg/mL) was better than the activity of controls and it matched withantibiofilm activity. The effect of extracts was better on G+ bacteria (MICs were < 78ñ625 µg/mL). For ethanoland ethyl acetate extracts all BIC values were better than MICs. Extracts showed a significant effect onAspergillus restrictus (MICs were < 78/156 µg/mL). The GC and GC-MS analysis of the ethyl acetate extractrevealed the identification of 22 compounds with (all E)-squalene (20.2%), n-alkanes and norlignanhinokiresinol among the most abundant ones. This is the first time that T. natans was studied using these meth-ods.

Keywords: Trapa natans, antimicrobial, antibiofilm, total phenolic content, flavonoid content, condensed tan-nins, volatile constituents

1565

* Corresponding author: e-mail: [email protected]; phone: +381 34 336 223

1566 IVANA D. RADOJEVI∆ et al.

only Trapa species occurring in Serbia was T.annosa, but T. natans is widely reported in the liter-ature as occurring in Serbia - the different state-ments almost certainly refer to the same taxon butonly in a few cases is it possible to establish whichtaxa are intended (7).

T. natans is an aquatic plant, located in stag-nant and slow flowing waters. The root is atrophied,the tree submerged, with long internodes, which areshorter near the water surface. Submerged leaves arefilar with early decline. Floating leaves form arosette. The leaves are serrated on a long petiole,with a central blister containing the air cavity. Theflowers in the axils of leaves are solitary and her-maphrodite (bipolar). It blooms from June toSeptember. The wooded fruit is of one seed with 2-4 stone fruit (2).

Relevant literature review has shown little dataabout the antimicrobial activity of T. natans leavesextracts. Several studies have been performed todemonstrate the antimicrobial effect of extracts pre-pared from different parts and the entire plant of T.natans (8-11). Mandal et al. (12) have tested peptidefrom T. natans fruits against Candida tropicalisbiofilm formation.

For this reason, the aim of the present study wasset to investigate antimicrobial and antibiofilm activ-ity of the extracts isolated from the leaves of this plantspecies applying in vitro methods; to determine thetotal phenol, flavonoid and tannin content from water,ethanol, acetone and ethyl acetate extracts, and toinvestigate if there is a connection between antimi-crobial activity and phytochemical content. Anothergoal of this study was to perform detailed composi-tional analysis of the T. natans volatile and semi-volatile compounds, since there are only few previousreports in the literature dealing with the volatile sec-ondary metabolites of this plant species (13-17).

EXPERIMENTAL

Chemicals

All chemicals were commercially availableand used as received. Nutrient liquid medium, aMueller-Hinton broth was purchased fromLiofilchem (Italy), while a Sabouraud dextrosebroth was from Torlak (Belgrade, Serbia). Anantibiotic, tetracycline was obtained from SigmaChemicals Co. (St. Louis, MO, USA) and antimy-cotic, fluconazole, was from Pfizer Inc. (USA).

Plant material

In July 2012, floating leaves of plant T. natanswere collected, in Me�uvröje Reservoir, central

Serbia (position: 43O51¥-43O56¥ N, 17O47¥-17O55¥ E,altitude: 273 m). Identification and classification ofthe plant material was performed at the Institute ofBiology and Ecology, Faculty of Science,University of Kragujevac. The voucher sample isdeposited at the Herbarium of the Department ofBiology and Ecology, Faculty of Science,University of Kragujevac (No. MB02/12). The col-lected plant material was air-dried in darkness atambient temperature. The dried plant material wascut up and stored in paper bags until needed.

Preparation of plant extracts

Dried, ground plant material was extracted bymaceration with solvents. The quantity of 50 g ofplant material was soaked with 250 mL of solvent.The plant material was macerated three times atroom temperature using fresh solvent every 24 h.The filtrates obtained were combined and then evap-orated to dryness using a rotary evaporator at 40OC,except for the water extract where a water bath wasused. The obtained extracts were kept in sterile sam-ple tubes and stored at -20OC.

In vitro antimicrobial assay

The antimicrobial activity of the plant extractswas tested against 26 microorganisms (15 strains ofbacteria and 11 strains of fungi). The list of testedmicroorganisms is presented in Table 1. All clinicalisolates were a generous gift from the Institute ofPublic Health, Kragujevac. The other microorgan-isms were provided from a collection held by theMicrobiology Laboratory, Faculty of Science,University of Kragujevac.

Bacterial and yeast suspensions were preparedby the direct colony method. The turbidity of initialsuspension was adjusted by comparing with 0.5McFarlandís standard (18). Initial bacterial suspen-sions contain about 108 colony forming units(CFU)/mL. 1 : 100 dilutions of initial suspensionwere additionally prepared into sterile 0.85% saline.The suspensions of fungal spores were prepared bygentle stripping of spore from agar slants with grow-ing aspergilli. The resulting suspensions were 1 :1000 diluted in sterile 0.85% saline.

Antimicrobial activity was tested by determin-ing the minimum inhibitory concentration (MIC) andminimum microbicidal concentration (MMC) usingmicrodilution method with resazurin (19). The stockconcentration of tested plant extracts was 10 000µg/mL. Twofold serial dilutions of plant extractswere made in sterile 96-well plates containingMueller-Hinton broth for bacteria and Sabourauddextrose broth for fungi. The tested concentration

Antimicrobial and antibiofilm effects of extracts from Trapa natans L... 1567

Tab

le 1

. Ant

ibac

teri

al a

ctiv

ity o

f w

ater

, eth

anol

, ace

tone

and

eth

yl a

ceta

te e

xtra

cts

of T

. nat

ans

agai

nst t

este

d m

icro

orga

nism

s ba

sed

on m

icro

dilu

tion

met

hod.

Spec

ies

Wat

er e

xtra

ctE

than

ol e

xtra

ctA

ceto

ne e

xtra

ctE

thyl

ace

tate

ext

ract

Tet

racy

clin

e

MIC

1M

MC

2M

ICM

MC

MIC

MM

CM

ICM

MC

MIC

MM

C

Bac

illus

sub

tilis

>500

0>5

000

<78

<78

<78

<78

<78

<78

0.5

0.5

Bac

illus

cer

eus

>500

0>5

000

313

2500

313

1250

313

1250

0.5

3.91

Bac

illus

pum

ilus

NC

TC

824

1>5

000

>500

062

525

0015

612

5062

525

000.

50.

5

Sarc

ina

lute

a>5

000

>500

0<7

831

3<7

862

5<7

812

50-

-

Stap

hylo

cocc

us a

ureu

s>5

000

>500

031

312

5031

362

531

312

500.

9815

.62

S. a

ureu

sPM

FKG

-B12

>500

0>5

000

<78

625

7862

5<7

862

50.

121.

95

S. a

ureu

sA

TC

C 2

5923

>500

0>5

000

313

625

313

625

313

1250

0.24

1.95

Ent

eroc

occu

s fa

ecal

isA

TC

C 2

9212

>500

0>5

000

2500

5000

2500

2500

2500

5000

7.81

62.5

Pse

udom

onas

aer

ugin

osa

>500

0>5

000

1250

5000

313

1250

2500

5000

500

1000

P. a

erug

inos

aA

TC

C 2

7853

>500

0>5

000

2500

5000

625

625

5000

>500

07.

8162

.5

Pro

teus

mir

abili

s>5

000

>500

031

350

0015

625

0012

5025

0050

050

0

P. m

irab

ilis

AT

CC

124

53>5

000

>500

012

5050

0015

662

525

0050

0012

512

5

Esc

heri

chia

col

iAT

CC

259

22>5

000

>500

050

00>5

000

313

5000

>500

0>5

000

0.98

3.91

Salm

onel

la e

nter

ica

>500

0>5

000

5000

5000

1250

2500

>500

0>5

000

1.95

3.91

Salm

onel

la ty

phim

uriu

m>5

000

>500

050

00>5

000

2500

2500

>500

0>5

000

0.98

3.91

1 MIC

val

ues

for

plan

t ext

ract

s an

d an

tibio

tic a

re g

iven

as

µg/m

L -

mea

ns in

hibi

tory

act

ivity

. 2 MM

C v

alue

s fo

r pl

ant e

xtra

cts

and

antib

iotic

are

giv

en a

s µg

/mL

- m

eans

mic

robi

cida

l act

ivity

.


range was from 5 000 to 78.13 µg/mL. Afterthat, the plates were inoculated with the suspen-sions to give a final concentration of 5 ◊ 105

CFU/mL for bacteria and 5 ◊ 103 CFU/mL forfungi. The growth of bacteria and yeasts wasmonitored by adding resazurin, an indicator ofmicrobial growth. The inoculated plates wereincubated at 37OC for 24 h for bacteria, 28OC for48 h for yeasts and 28OC for 72 h for molds.MIC was defined as the lowest concentration oftested substance that prevented resazurin colorchange from blue to pink. For molds, MIC val-ues of the tested substance were determined asthe lowest concentration that visibly inhibitedmycelia growth. MMC was determined by plat-ing 10 mL of samples from wells, where noindicator color change was recorded, on nutrientagar medium. At the end of the incubation peri-od the lowest concentration with no growth (nocolony) was defined as MMC. Tetracycline andfluconazole, dissolved in nutrient liquid medi-um, were used as positive controls. Stock solu-tions of crude extracts were obtained by dis-solving in 10% DMSO which was used as acontrol. It was observed that 10% DMSO didnot inhibit the growth of microorganism. Eachtest included growth control and sterility con-trol. All tests were performed in duplicate andMICs were constant.

Tissue culture plate method (TCP)

The TCP assay described by Christensenet al. (20) is most widely used test for detectionof biofilm formation. We screened all strainsfor their ability to form biofilm by TCP methodwith some modifications. Each test includedbiofilm formation control. Bacterial biofilmformation properties were well described byOíToole et al. (21). Anti-biofilm activity wascalculated in form of the biofilm inhibitoryconcentration (BIC) which is the concentrationthat showed inhibition at different percentages.The tissue culture 96-well plates (Sarstedt)were prepared by dispensing 50 µL of nutrientbroth, Mueller-Hinton broth for bacteria, intoeach well. A 50 µL from the stock solution oftested extracts (concentration of 20 000 µg/mL)was added into the first row of the plate. Then,twofold, serial dilutions were performed byusing a multichannel pipette. A 50 µL of freshbacterial suspension was added to each well.Initial bacterial suspensions contain about 108

CFU/mL. Dilutions of initial suspension (1 :100) were additionally prepared into Mueller-

Tab

le 2

. Ant

ifun

gal a

ctiv

ity o

f w

ater

, eth

anol

, ace

tone

and

eth

yl a

ceta

te e

xtra

cts

of T

. nat

ans

agai

nst t

este

d m

icro

orga

nism

s ba

sed

on m

icro

dilu

tion

met

hod.

Spec

ies

Wat

er e

xtra

ctE

than

ol e

xtra

ctA

ceto

ne e

xtra

ctE

thyl

ace

tate

ext

ract

Tet

racy

clin

e

MIC

1M

MC

2M

ICM

MC

MIC

MM

CM

ICM

MC

MIC

MM

C

Rho

doto

rula

sp.

5000

>500

012

5025

0012

5025

0025

0050

0062

.50

1000

Can

dida

alb

ican

s25

00>5

000

5000

>500

012

5025

0050

00>5

000

62.5

010

00

Can

dida

alb

ican

sAT

TC

102

3150

00>5

000

5000

>500

025

0050

0050

00>5

000

31.2

510

00

Sacc

haro

myc

es b

oula

rdii

>500

0>5

000

>500

0>5

000

>500

0>5

000

>500

0>5

000

31.2

510

00

Pen

icill

ium

dig

itatu

m25

0050

0031

362

525

0050

0012

5025

0031

.25

31.2

5

P. v

eruc

osum

2500

5000

<78

2500

156

156

156

156

--

Asp

ergi

llus

rest

rict

us50

0050

0015

615

6<7

850

00<7

8<7

850

020

00

Asp

ergi

llus

flavu

s50

00>5

000

5000

>500

0>5

000

>500

025

0050

0010

0010

00

Asp

ergi

llus

nige

r50

00>5

000

5000

>500

025

00>5

000

2500

>500

050

010

00

A.

nige

rAT

CC

164

0450

00>5

000

1250

>500

050

00>5

000

1250

>500

062

.562

.5

Bot

rytis

cyn

erea

2500

>500

050

00>5

000

5000

>500

050

00>5

000

31.2

550

0

1 MIC

val

ues

for

plan

t ext

ract

s an

d an

tibio

tic a

re g

iven

as

µg/m

L -

mea

ns in

hibi

tory

act

ivity

. 2 MM

C v

alue

s fo

r pl

ant e

xtra

cts

and

antib

iotic

are

giv

en a

s µg

/mL

- m

eans

mic

robi

cida

l act

ivity

.


Hinton broth. The obtained extract concentrationrange was from 10 000 to 78 µg/mL. The inoculatedplates were incubated at 37OC for 24 h. After incu-bation, the content of each well was gently removedby tapping the plates. The wells were washed with200 µL of sterile 0.85% saline to remove free-float-ing bacteria. Biofilms formed by adherent cells inplate were stained with crystal violet (0.1% w/v) andincubated at the room temperature for 20 min.Excess stain was rinsed off thorough washing withdeionized water and plates were fixed with 200 µLof ethanol. Optical densities (OD) of stained adher-ent bacteria were determined with a micro ELISAplate reader at wavelength of 630 nm (OD630 nm).Only broth or broth with extracts served as controlto check sterility and non-specific binding of media.To compensate for background absorbance, ODreadings from sterile medium, extracts, fixative anddye were averaged and subtracted from all test val-ues.

Biochemical analysis

Determination of total phenolic content The total phenolic content was determined by

using Folin-Ciocalteuís method (22). The reactionmixture was prepared by mixing 0.2 mL ofmethanolic solution of extract (1 mg/mL) and 1.5mL of 1 : 10 Folin-Ciocalteuís reagent dissolved inwater. The mixture was allowed to equilibrate for 5min and then mixed with 1.5 mL 6% Na2CO3 solu-tion. After incubation for 90 min at room tempera-ture in darkness, the absorbance of the mixture wasread at 725 nm against a blank using spectropho-tometer. The blank was prepared with methanolinstead of extract solution. The samples were pre-pared in triplicate and the mean value of absorbancewas obtained. The same procedure was repeated forgallic acid which was used for calibration of stan-dard curve. Total phenol content is reported as gal-lic acid equivalents by reference to linear equationof the standard curve (y = 0.008x + 0.0077, R2 =0.998). Then, the total phenolic content wasexpressed as a milligram of gallic acid equivalentper gram of extract (mg GAE/g of extract).

Determination of total flavonoid content The concentrations of flavonoids were deter-

mined by using aluminium chloride method (23).The mixture contained 2 mL of methanolic solutionof extract (1 mg/mL) and 2 mL of 2% methanolicAlCl3◊6H2O solution. The mixture was vigorouslyshaken, and after 10 min of incubation at room tem-perature, the absorbance versus a prepared blank wasread at 430 nm using spectrophotometer. The sam-

Tab

le 3

. Ant

ibio

film

act

ivity

of

etha

nol,

acet

one

and

ethy

l ace

tate

ext

ract

s of

T. n

atan

s ag

ains

t tes

ted

mic

roor

gani

sms.

Spec

ies

Eth

anol

ext

ract

A

ceto

ne e

xtra

ct

Eth

yl a

ceta

te e

xtra

ct

Tet

racy

clin

e

BIC

50o

BIC

90o

BIC

50o

BIC

90o

BIC

50o

BIC

90o

BIC

50o

BIC

90o

Esc

heri

chia

col

i AT

CC

259

2226

4549

3330

5554

3986

411

8736

125

Pro

teus

mir

abili

s18

6841

2416

9017

9811

0025

5042

666

Pse

udom

onas

aer

ugin

osa

1703

3567

/92

315

3039

9574

620

00

P. a

erug

inos

aA

TC

C27

853

//

720

1171

1414

2356

156

305

o Min

imum

bio

film

inhi

bitio

n co

ncen

trat

ion

for

plan

t ext

ract

s an

d an

tibio

tic a

re g

iven

as

µg/m

L ñ

mea

ns 5

0% in

hibi

tion

on th

e bi

ofilm

for

mat

ion;

o Min

imum

bio

film

inhi

bitio

n co

ncen

trat

ion

for

plan

t ext

ract

s an

dan

tibio

tic a

re g

iven

as

µg/m

L ñ

mea

ns 9

0% in

hibi

tion

on th

e bi

ofilm

for

mat

ion;

/ m

eans

not

det

erm

ined

.


ples were prepared in triplicate and the mean value ofabsorbance was obtained. Rutin was used as a stan-dard for calibration of standard curve. The concen-trations of flavonoids were calculated from the linearequation of standard curve (y = 0.021x + 0.040, R2 =0.999). Then the concentrations of flavonoids wereexpressed as milligram of rutin equivalent per gramof extract (mg of RU/g of extract).

Determination of condensed tannins (proantho-cyanidins)

Condensed tannins were determined using thebutanol HCl method (24). The reaction mixture wasprepared by mixing 0.5 mL of methanolic solutionof extract (1 mg/mL) with 3 mL of the butanol-HClreagent (butanol-HCl 95 : 5, v/v) and 0.1 mL of theferric reagent (2% ferric ammonium sulfate in 2MHCl). The covered tubes were heated in a water bathat 100OC for 60 min. After cooling, the absorbanceversus a prepared blank was read at 550 nm usingspectrophotometer. The samples were prepared intriplicate and the mean value of absorbance wasobtained. Cyanidin chloride was used as a standardfor calibration of standard curve. The concentrationsof proanthocyanidins were calculated from the lin-ear equation of standard curve (y = 0.0094x + 0.006,R2 = 0.999). Then, the concentrations of proantho-cyanidins were expressed as milligram of cyanidinchloride equivalent per gram of extract (mg ofCChE/g of extract).

GC-FID and GC-MS analyses

Chemical composition of the ethyl acetateextract was investigated by GC and GC-MS. TheGC-MS analyses were performed in triplicate on aHewlett-Packard 6890N gas chromatographequipped with a DB-5 phenyl methyl siloxane capil-lary column (30 m ◊ 0.25 mm, film thickness 0.25µm, Agilent Technologies, USA) and coupled witha 5975C mass selective detector from the same com-

pany. The injector and interface were operated at250 and 320OC, respectively. Oven temperature wasraised from 70 to 315OC at a heating rate of 5OC/minand then isothermally held for 10 min. As a carriergas, helium at 1.0 mL/min was used. The samples (1µL of the extract solution) were injected in a pulsedsplit mode (split ratio 40 : 1). The MS conditionswere as follows: ionization energy 70 eV, acquisi-tion mass range 35ñ650 amu and scan time 0.34 s.GC-FID analysis was carried out under the sameexperimental conditions using the same column asdescribed for the GC-MS.

Qualitative and quantitative analysis

The volatile and semivolatile constituents wereidentified by comparison of the their linear retentionindices (determined relative to the retention times ofC8ñC32 n-alkanes on the DB-5 column (25), to thosereported in the literature (26), and by comparison oftheir mass spectra to those of authentic standards, aswell as those from Wiley 6, NIST05 andMassFinder 2.3 libraries. Also, a homemade MSlibrary with the spectra corresponding to pure sub-stances was used, and finally, wherever possible, theidentification was achieved by coinjection with anauthentic sample. The percentage composition wascomputed from the GC-FID peak areas without theuse of correction factors.

Data analysis

All data were presented as the means ± stan-dard deviations (mean ± SD) where appropriate. Allstatistical analyses were performed using MicrosoftExcel software.

RESULTS


The results of in vitro antimicrobial activity offour extracts from T. natans leaves against 26 strains

Table 4. Yield, concentration of total phenolic, flavonoid content and condensed tannins in the extracts of T. natans.

Total phenolic Flavonoid CondensedType of Yield contenta contenta tanninsa

extract (g/50 g d.w.) (mg GA/g of (mg RU/g of (mg CChE/g of extract) extract) extract)

Water 1.58 g 39.44 ± 0.05 2.67 ± 0.11 2.52 ± 0.11

Ethanol 1.51 g 63.49 ± 0.15 79.72 ± 0.36 7.55 ± 0.02

Acetone 0.83 g 164.47 ± 0.83 91.98 ± 0.66 7.92 ± 0.13

Ethyl acetate 0.55 g 37.55 ± 0.14 63.69 ± 0.28 8.36 ± 0.03

a Each value shown is the mean value ± standard deviation.


of bacteria and fungi, with control results, deter-mined by microdilution method, are presented inTables 1 and 2. The intensity of antimicrobial actionvaried depending on the type of plant extract and onthe groups of microorganisms. MICs and MMCsvalues were in range from < 78.13 to > 5000 µg/mL. Water extract didnít show antibacterial activity(MIC/MMC were > 5000 µg/mL). Antifungal activ-ity of this extract was slightly better and hadnít gonebelow 2500 µg/mL. The acetone extract showed thestrongest antibacterial activity. Ethanol and ethylacetate extracts had a moderate effect mostly on G+bacteria. The exception is the ethanol extract on the

species Proteus mirabilis, where the MIC was at 313µg/mL and which acted better than the positive con-trol.

G+ bacteria were more sensitive than G- bacte-ria. MICs were in range < 78 ñ 625 µg/mL. Anexception is the species Enterococcus faecalisATCC 29212 at which the MIC for three extractswas 2500 µg/mL. But even if the effect of extractswas better on G+ bacteria it was here better than thecontrol tetracycline. At G- bacteria acetone andethanol extracts acted on P. mirabilis better than thecontrols (MICs were 313/156 µg/mL) and acetoneon Pseudomonas aeruginosa (MIC was 313 µg/mL).

Table 5. Volatile and semivolatile constituents of T. natans ethyl acetate extract.

RI calc.a Compound Content [%]b Identification method

1100 Undecane 2.5 RI, MS, Co-GC

1200 Dodecane t RI, MS, Co-GC

1700 Heptadecane t RI, MS, Co-GC

1800 Octadecane t RI, MS, Co-GC

1842 Neophytadiene (isomer I) 3.3 RI, MS

1846 Hexahydrofarnesyl acetone t RI, MS

1866 Neophytadiene (isomer II) t RI, MS

1884 Neophytadiene (isomer III) 4.9 RI, MS

1900 Nonadecane t RI, MS, Co-GC

1996 Ethyl hexadecanoate 1.5 RI, MS, Co-GC

2086 1-Octadecanol 1.4 RI, MS, Co-GC

2117 (E)-Phytol 7.0 RI, MS, Co-GC

2220 (E)-Phytyl acetate 4.3 RI, MS

2300 Tricosane t RI, MS, Co-GC

2307 Unidentified constituentc 3.3

2353 5-Methyl-5-(4,8,12-trimethyltridecyl) 1.2 RI, MSdihydro-2(3H)-furanone

2396 Hinokiresinold 9.2 MS

2500 Pentacosane 4.0 RI, MS, Co-GC

2700 Heptacosane 13.1 RI, MS, Co-GC

2800 Octacosane 12.4 RI, MS, Co-GC

2833 (All E)-Squalene 20.2 RI, MS

2900 Nonacosane t RI, MS, Co-GC

3143 α-Tocopherol 5.3 RI, MS, Co-GC

Identified 90.3

Unidentified (> 0.5%) 3.3

Total 93.6

a Linear retention indices experimentally determined on the DB-5 column; b Values are means of three individual analyses; c MS, 70 eV,230OC, m/z (rel. int.): 246 (7), 124 (7), 123 (8), 122 (7), 105 (6), 98 (7), 97 (100), 69 (9), 55 (8), 41 (10); d Correct isomer was not deter-mined; RI - Retention indices matching with literature data; MS - mass spectra matching; Co-GC - co-injection with pure reference com-pound; t - Trace amounts (< 0.05%).


If comparing with positive control, the testedextracts showed low antifungal activity. Ethanol,acetone and ethyl acetate extracts showed a signifi-cant effect on species Aspergillus restrictus wherethe MICs were < 78/156 µg/mL and better than flu-conazole. The most resistant species wasSaccharomyces boulardii in which MIC and MMCexceeds everywhere 5000 µg/mL.

Antibiofilm activity

Three different extracts of this plant were cho-sen for anti-biofilm testing (Table 3). All of themshowed different potential depending on bacterialstrain and type of extract. The analyses showed thatthe percentage of the reduction of biofilm formationand concentrations of used extracts were in expo-nential dependence, so that under the certain extractconcentrations there was no any influence. The bestactivity had acetone extract, with the most sensitivecase where BIC90 for P. aeruginosa was 923 µg/mL.The result matches with antibacterial activity, wherethe same extract acted the strongest against P. aerug-inosa (MIC at 313 µg/mL and MMC at 1250µg/mL). Acetone extract had overall the best anti-biofilm effect. The less sensitive was E. coli ATCC25922 at all the tested extracts. In that case all BICvalues are lower than MICs for ethanol and ethylacetate extracts and also for ethyl acetate extractagainst P. aeruginosa ATCC 27853. The BIC90 val-ues for all the other tested combinations werenoticeable higher than the MIC values.

Total phenol, flavonoid content and condensed

tannins

The percent yields of crude extracts, concen-tration of total phenolic, flavonoid and condensedtannins obtained from T. natans are presented inTable 4. Water extracts had the greatest yield, buthad a noticeably lower amount of total phenolics,flavonoids and condensed tannins. Acetone extracthad in itself a significantly greater amount of totalphenolics and flavonoids.

Chemical composition of the volatile and semi-

volatile constituents

Table 5 lists the identified volatile and semi-volatile constituents of T. natans ethyl acetate extractby means of a detailed GC and GC/MS. The analy-ses allowed the identification of 22 compounds, with(all E)-squalene (20.2%), heptacosane (13.1%) andoctacosane (12.4) as the most abundant ones. Aboutone-third of the composition was comprised of n-alkanes. It is interesting to note that, beside the ubiq-uitous components of extracts, GC-MS showed the

presence of norlignan (cis- or trans-) hinokiresinol inthe ethyl acetate extract of T. natans.

DISCUSSION

In previous research, the antimicrobial activityof different parts of the plant T. natans showed dif-ferent results. In accordance with our study, waterextract seemed to be less efficient to inhibit thegrowth of microorganisms compared to other testedextracts (8). To obtain new results, the wide range ofmicroorganisms tested have been chosen to showthe potential of T. natans leaf extracts against foodspoiling strains, clinical isolates, biofilm formingbacteria and standard strains.

Acetone extract isolated from T. natans leaveshave important phytochemicals such as phenoliccompounds and tannins. This extract showed maxi-mum antimicrobial activity against bacterial andfungal strains (11). In this study the acetone extractacted on P. aeruginosa better or in the range of thepositive control. This extract acted also on other G-bacteria better than other extracts, which can be con-nected with the fact that it also had a significantlyhigher total phenol and flavonoid content. The pres-ent compounds may be responsible for the medicinalproperties of this plant (11).

Stoicescu et al. (10) was evaluating the antibac-terial activity of ethanolic extracts from all compo-nent parts of water chestnuts. Although the leafextract (aerial plant parts) had the highest polyphenolcontent, it didnít show antibacterial activity. The rea-son is that the method used was disc diffusion andthat start testing concentrations were low (0.03 to0.15 µg/mL) lower than antibiotic concentrations. Atthose concentrations, only the samples obtained fromthe fruit pericarp presented an antibacterial effect onStaphylococcus aureus and Escherichia coli.

Mandal et al. (12) tested peptide from T. natansfruits against Candida tropicalis biofilm formation.They found that peptide Tn-AFP1 had influencedbiofilm of this yeast and also tested biofilm regula-tive genes of C. tropicalis on real-time PCR. Up tonow, there has been no data about this plantís influ-ence on bacterial biofilm.

Previous phytochemical investigation of theethanol extract isolated from T. natans rootsrevealed the presence of flavonoids, carbohydrates,phenols, tannins, sterols, proteins and fats (27).Ferulic and caffeic acid were also detected (27).Aqueous extract of T. natans L. fruits rind wasfound to contain a large amount of polyphenols (28).

The chemical analysis of the ethyl acetateextract revealed the presence of norlignan


hinokiresinol. cis-Hinokiresinol was found to showantioxidant and antiatherogenic activities (29). Thiscompound, also known as nyasol, was recently iso-lated from a closely related species of the samegenus, an aquatic herbaceous plant, T. pseudoincisa(29). Nyasol was also found in the plant speciesAnemarrhena asphodeloides, Chamaecyparis obtusaand Araucaria angustifolia (30-33). This compoundshowed antimicrobial activity against fungi and bac-teria with MIC values in a range of 12.5 to 400µg/mL (33). Since these plants are used in tradition-al medicine, it is expected for nyasol to be less toxicto humans (33).

Squalene is a triterpene and has been reportedto have antimicrobial activity (34). Natural squaleneexhibited potent antimicrobial activity. It inhibitedthe growth of Sarcina lutea and Escherichia coli andalso retarded the growth of fungi causing aspergillo-sis (35).

CONCLUSIONS

The acetone extract of T. natans leaves is animportant source of biologically active compoundsand thus shows potential in the control of microor-ganisms. Antimicrobial activity was expressed espe-cially on the tested bacteria. This effect was signifi-cant on G- bacteria, especially against Pseudomonasaeruginosa, where the observed effect was betterthan the control, both at the level of inhibition ofgrowth (microdilution activity) and the level ofreduction of biofilm formation. The activity that theextracts showed, especially the acetone one, implythat they can be used, after further investigation, asa natural source of antimicrobials preventing thegrowth of pathogenic and food spoilage microor-ganisms, in the pharmaceutical industry, naturaltherapies and for improvement of human health.

Acknowledgments

This investigation was supported by theMinistry of Education, Science and TechnologicalDevelopment of the Republic of Serbia, grants No.III 41010, OI 173032 and OI 172061.

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2. JankoviÊ M.M.: in Flora SR Srbije (in Serbian),JosifoviÊ M. Ed., pp. 35ñ46. SANU, Beograd1973.

3. JankoviÊ M.M.: Ecology, distribution, taxono-my and history of the genus Trapa L. inYugoslavia. (in Serbian). p. 143, SerbianBiological Society, Beograd 1958.

4. Stevens P.F.: Angiosperm Phylogeny WebsiteVersion 5, May 2004. http://www.mobot.org/MOBOT/research/APweb/, 2001 (accessed 14.March 2005).

5. Porcher M.H. et al.: Sorting Trapa names.Multilingual Multiscript Plant Name DatabaseñA,work in progress. Institute for Land and FoodResources, The University of Melbourne.ttp://www.plantnames.unimelb.edu.au/ Sorting/Trapa.html 1995 (accessed 30. May 2009).

6. JankoviÊ M., BlaûenËiÊ J.: in The Red DataBook of Flora of Serbia l. Extinct and CriticallyEndangered taxa (In Serbian), StevanoviÊ V.Ed., pp. 54-56. Publication of Ministry of envi-ronment of Republic of Serbia, Faculty ofBiology, University of Belgrade & Institutionfor Protection of Nature of the Republic ofSerbia, Beograd 1999.

7. Lansdown R.: Trapa natans. In IUCN 2013.IUCN Red List of Threatened Species. Version2013.2. www.iucnredlist.org 2011 (accessed 22May 2014).

8. Parekh J., Chanda S.: Afr. J. Biotechnol. 6, 766(2007).

9. Agarwal T., Shukla D.A., Gujrati A., SrivastavaN.: Int. J. Pharm. Res. Dev. 3/4, 114 (2012).

10. Stoicescu I., Sirbu R., Negreanu-Pirjol T.,Cociasu M., Balaban D.P. Bala C.: Rev. Roum.Chim. 57, 729 (2012).

11. Kumar D., Rashid M., Singh A.P.: Int. J. Pharm.Pharm. Sci. 3, 1697 (2014).

12. Mandal S.M., Migliolo L., Franco O.L., GhoshA.K.: Peptides 32, 1741 (2011).

13. Kameoka H., Kishi H.: Nippon Nogei Kaishi53, 57 (1979).

14. Kameoka H., Kishi H., Yakugaku Z.: J. Pharm.Soc. Japan 100, 114 (1980).

15. Liang R., Peng Q.J.: Zhong Yao Cai 29, 24(2006).

16. Zhao W., Niu F., Li J., Dong Q., Huang Z.:Chem. Res. Chin. Univ. 25, 56 (2009).

17. Li J., Xu W., Niu F.: Zhongguo Zhong Yao ZaZhi 36, 1725 (2011).

18. Andrews J.M.: J. Antimicrob. Chemother. 56,60 (2005).

19. Sarker S.D., Nahar L., Kumarasamy Y.:Methods 42, 321 (2007).

20. Christensen G.D., Simpson W.A., Younger J.J.,Baddour L.M., Barrett F.F. et al., J. Clin.Microbiol. 22, 996 (1985).


21. OíToole G., Kaplan H.B., Kolter R.: Annu.Rev. Microbiol. 54, 49 (2000).

22. Wootton-Beard P.C., Moran A., Ryan L.: FoodRes. Int. 44, 217 (2011).

23. Quettier-Deleu C., Gressier B., Vasseur J., DineT., Brunet C. et al.: J. Ethnopharmacol. 72, 35(2000).

24. Porter L.J., Hrstich L.N., Chan B.G.:Phytochemistry 25, 223 (1986).

25. Van den Dool H., Kratz P.D.: J. Chromatogr. A11, 463 (1963).

26. Adams R.P.: Identification of Essential OilComponents by Gas Chromatography/MassSpectrometry, p. 804, Allured Publishing Corp.,Carol Stream, IL 2007.

27. Kharbanda C., Sarwar Alam M., Hamid H.,Bano S., Haider S. et al.: J. Ethnopharmacol.151, 931 (2014).

28. Malviya N., Jain S., Jain A., Jain S., Gurjar R.:Acta Pol. Pharm. Drug Res. 67, 391 (2010).

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Received: 5. 10. 2015


Diabetes mellitus is usually described as a dis-ease associated with chronically raised blood glu-cose levels. In fact, it is a complex disease charac-terized by absolute insulin deficiency in the case oftype I diabetes and relative insulin deficiency withor without insulin resistance in type II diabetes. Itassumes the form of a complex syndrome over itschronic course, with wide derangements in carbohy-drate, lipid and protein metabolism and is associatedwith life threatening complications. The chronichyperglycemia is the common feature of all hetero-geneous disorders grouped under diabetes mellitus(1). Long term elevated blood glucose levels canlead to increased circulating levels of advanced gly-cation end products (AGEs) causing vascular com-plications that may ultimately lead to diabeticretinopathy, nephropathy, neuropathy, and increasethe risk of stroke and myocardial infarction (2).Maintenance of normal blood glucose levels is cru-

cial to avoid the synthesis of AGEs and delay vas-cular complications. The currently used antidiabeticdrugs are associated with significant adverse effectsand there is still a need to search for medicinalplants with therapeutic potential.

Medicinal plants have been traditionally usedas a source of medicine in virtually all cultures (3).The global interest has recently increased in non-synthetic and natural medicines derived from plantsources due to their better tolerance and minimumadverse effects. According to the WHO estimates,about three-quarters of the worldís population cur-rently use herbs and other forms of traditional med-icines for the treatment of various diseases (4).Ethnopharmacological surveys indicate that morethan 1200 plants are used traditionally for theiralleged hypoglycemic activity (3). There are around410 experimentally proven medicinal plants thathave antidiabetic properties, but the complete mech-

ANTIDIABETIC AND ANTIDYSLIPIDEMIC EFFECTS OF HELIOTROPIUMSTRIGOSUM IN RAT MODELS OF TYPE I AND TYPE II DIABETES

SHAFQAT RASUL CHAUDHRY1,2*, ADNAN AKRAM1, NAVEED ASLAM1, MUHAMMAD ASIF1,MUHAMMAD WAJID1, THOMAS KINFE2, QAISER JABEEN1 and SAJJAD MUHAMMAD2

1Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan2Department of Neurosurgery, University Hospital Bonn, University of Bonn, Bonn 53105, Germany

Abstract: Heliotropium strigosum Willd. (Boraginaceae) is used traditionally as a laxative, diuretic, and as atreatment for snake bites and stings of nettles. Recent investigations have shown anti-inflammatory and antioxi-dant activity of H. strigosum. However, antihyperglycemic and antidyslipidemic activity of H. strigosum has notbeen investigated to date and we aimed to explore these activities of the crude aqueous methanolic extract of theaerial parts of H. strigosum (Hs.Cr). Hs.Cr was administered orally at doses of 100, 300, and 500 mg/kg in allox-an-induced diabetic rats (type I diabetes) and fructose-fed rats (type II diabetes). The fasting blood glucose (FBG)concentration was assessed by glucometer, while serum total cholesterol, triglycerides and HDL were estimatedby using standard kits. The FBG concentration significantly (p < 0.05) decreased in dose-dependent pattern inboth alloxan-induced diabetic and fructose-fed rats on Hs.Cr administration. The percentage glucose reductionsin alloxanized rats with glibenclamide, Hs.Cr 100, 300, and 500 mg/kg were obeserved to be 67, 36, 56 and 62%,respectively. In fructose-fed rats, the percentage glucose redutions associated with metformin, Hs.Cr 100, 300,and 500 mg/kg were 23, 5, 11 and 12%, respectively. The extract also corrected the dyslipidemia associated withfructose and alloxan-induced diabetes by significantly (p < 0.001) decreasing the concentration of serum totalcholesterol, triglycerides and LDL and by increasing HDL concentration. Our data demonstrate that the H. strigo-sum has antidiabetic and antidyslipidemic effects, thus encouraging further studies.

Keywords: antihyperglycemia, hypoglycemia, alloxan, antihyperlipidemia, hypolipidemia, fructose-fed dia-betes model, insulin resistance, metabolic syndrome

1575

* Corresponding author: e-mail: [email protected]; phone: 0049-228-287-16448; address: Sigmund-Freud-Strasse 25, 53105Bonn, Germany

1576 SHAFQAT RASUL CHAUDHRY et al.

anism of action is available only for about 109medicinal plants (5).

Heliotropium strigosum Willd. (Boraginaceae)(English: Indian Forget-me-not, locally known asGorakh pan or Chittiphul) is traditionally used as adiuretic and laxative (6ñ8). The plant juice is appliedto gum boils, sore eyes and sores, generally with theintention to promote suppuration. It is employed asa cure for stings of nettles, insects, and snake bites(6ñ12). The crude plant extract and its various frac-tions have been shown to possess antioxidant andantimicrobial activities (13). Anti-inflammatory,anti-nociceptive and anticonvulsant activities havealso been reported (14, 15). A phthalic acid esterisolated from chloroform fraction has also showncompetitive trypsin inhibition (16).

Heliotropium zeylanicum, a closely relatedspecies to H. strigosum, has been evaluated forantidiabetic, antihyperlipidemic and antioxidanteffects in streptozotocin induced diabetic rats (17).H. strigosum has also been found to possess antiox-idant properties (13). Many studies have shown thatplants bearing antioxidant properties also have goodantidiabetic activity (18ñ20). Therefore, the aim ofthe current study was to investigate the antidiabeticand antidyslipidemic effects of the H. strigosum inanimal models of type I and type II diabetes.


Preparation of the crude extract

The dried aerial-parts of the plant, purchasedfrom a herbal shop in Bahawalpur, were authenticat-ed by a taxonomist. A specimen of plant materialwas preserved in the herbarium of the Faculty ofPharmacy and Alternative Medicine of the IslamiaUniversity of Bahawalpur in Pakistan and a voucherno. HS-AP-04-11-018 was assigned for future refer-ence. The clean plant material was ground to coarsepowder (approximately 920 g) and was then macer-ated in 70% aqueous methanol. After three days ofmaceration, it was filtered through muslin cloth andsubsequently through Whatman filter paper no. 1.The filtrate was preserved, while residue was recy-cled twice to obtain more filtrate. Finally, the pooledfiltrate from the three macerations was evaporated ona rotary evaporator (Heidolph Laborota 4000 effi-cient, Germany) at 30-40OC. A concentrated, thickviscous material with semisolid consistency and darkbrown color was obtained with a yield of 6.6%. Theextract was labelled as Hs.Cr and it was preserved inan air tight container at -20OC until further use. TheHs.Cr was resuspended in normal saline to preparedifferent doses prior to administration (21).

Preliminary phytochemical analysis

The preliminary phytochemical screening wasperformed as described by Gilani et al., (22).Briefly, alkaloids were detected by employingDragendroffís reagent and Mayerís reagent, whilesaponins were detected by froth test. Treatment of abenzene fraction of the Hs.Cr with ammonia toyield a pink, violet, and red coloration was used todetect the presence of anthraquinones. The filterpaper impregnated with dilute sodium hydroxidewas exposed to the boiling extract solution andvisualized for yellow fluorescence to detectcoumarins. The defatted Hs.Cr was dissolved in80% ethanol and then treated with 1% methanolicaluminium chloride and 1% potassium hydroxideseparately to detect flavanols, flavones, and/or chal-cones. The generation of intense green, purple,blue, and black color upon treatment with 1% aque-ous or alcohlic ferric cholride indicated the pres-ence of tannins. Keller-Kiliani test was used todetect glycosides.

Experimental animals

Albino Sprague-Dawley rats of both sexes,weighing 150-200 g and Swiss albino mice of bothsexes (18-32 g), housed in the animal facility ofthe pharmacology section of the Faculty ofPharmacy and Alternative Medicine of the IslamiaUniversity of Bahawalpur in Pakistan, were usedin the study unless otherwise stated. The standardconditions of temperature (25 ± 2OC) and humidi-ty (45-55%) along with exposure to a 12 : 12 hlight and dark cycle were maintained throughoutthe study. Animals were fed with a normal animaldiet and allowed to drink water ad libitum.Animals were acclimatized to the experimentalconditions for one week before starting the studyto minimize animal stress. The study protocols andprocedures were approved by the local animalethics committee.

Acute toxicity assessment

Albino mice were randomly divided into fivedifferent groups with six mice in each. Fourgroups received the crude extract orally by gavagein the order of increasing doses of 100, 300, 1000,and 3000 mg/kg, respectively, for each group. Acontrol group receiving normal saline (10 mL/kg)was run parallel to the extract receiving groups.The mice were given food and water ad libitum.The mice were observed regularly during the first6 h for any behavioral changes. The lethal effectswere assessed after 24 and 48 h of extract admin-istration (22).

Antidiabetic and antidyslipidemic effects of Heliotropium strigosum... 1577

Hypoglycemic effect in normoglycemic rats

The acute hypoglycemic effect of the Hs.Crwas evaluated in male rats weighing 250-350 g.Animals were randomized into different groups withsix rats in each. Overnight fasted (12 h) rats weregiven extract in the order of increasing dose i.e.,100, 300 and 500 mg/kg, respectively, for eachgroup. The normal control group received normalsaline only and a standard control group receivedglibenclamide (Daonil, Sanofi-Aventis, Pakistan) atthe dose of 5 mg/kg. Fasting blood glucose (FBG)concentration was determined at 0 h (prior to extractadministration), 1, 2, 4 and 6 h intervals with thehelp of single touch glucometer (Accu-chek Active,Roche, Germany) from the rat tail vein blood(23ñ25). Normal fasting rats received the respectivetreatments according to the following experimentaldesign:

Group I: Normal control (Normal saline 1mL/kg);

Group II: Standard control (Glibenclamide5 mg/kg);

Group III: Hs.Cr 100 mg/kg;Group IV: Hs.Cr 300 mg/kg;Group V: Hs.Cr 500 mg/kg.

Alloxan-induced diabetes mellitus

Alloxan monohydrate (Acros Organics, USA)at the dose of 140 mg/kg (26, 27) was dissolved innormal saline and was injected intraperitoneally tothe 18 h fasted rats (28, 29). This dose of alloxanwas standardized by previous pilot studies. Alloxanwas freshly prepared just prior to the injection in icecold normal saline and kept on ice bath while inject-ing the rats. A dextrose solution (20% w/v) wasadministered after 6 h to prevent the rats from allox-an induced hypoglycemia due to massive release ofinsulin from pancreas (30). For the next 24 h, ratswere maintained on 5% glucose in drinking water(25). Diabetes was confirmed after 72 h of injectionby using glucometer and the animals with a fastingblood glucose level ≥ 200 mg/dL were included inthe study.

The rats were allowed to stabilize until day 7post injection. On the 7th day, animals were random-ized into six groups and their fasting blood glucoselevels were assessed. The treatment was initiated onthe same day, considering it the 1st day of the study(25, 31, 32). Further assessment of FBG concentra-tion was carried out on 3rd, 7th, and 14th day of thestudy, respectively. The treatment protocol followedis given as below:


Group II: Positive control (Alloxan 140mg/kg + Normal saline 1mL/kg);

Group III: Standard control (Alloxan 140mg/kg + Glibenclamide 10mg/kg);

Group IV: Hs.Cr 100 mg/kg (Alloxan 140mg/kg + Hs.Cr 100 mg/kg);

Group V: Hs.Cr 300 mg/kg (Alloxan 140mg/kg + Hs.Cr 300 mg/kg);

Group VI: Hs.Cr 500 mg/kg (Alloxan 140mg/kg + Hs.Cr 500 mg/kg).

High fructose-fed model

Albino rats were randomly divided into sixgroups, with each group containing six animals.Animals in high fructose-fed groups were receiving60% fructose (BDH, England) in their diet (33, 34)as compared to normal control group, receiving nor-mal diet in which 60% w/w fructose was replacedwith vegetable starch. The FBG concentration wasdetermined at the start of treatment and then onweekly basis. After completing 6 weeks of treatmentaccording to the designed experimental protocol,animals were sacrificed and blood was withdrawnby cardiac puncture to obtain lipid profile (33).

One day before sacrificing, a glucose tolerancetest was conducted by administering 30% solutionof glucose at the dose of 2 g/kg in overnight fastedrats (35). Glucometer was used to check the bloodglucose concentration at 0, 30, 60, and 120 min fromthe tail vein of the rats.

The following treatment protocol was fol-lowed:

Group I: Normal control (Normal diet +Normal saline 1 mL/kg);

Group II: Positive control (High fructosediet + Normal saline 1 ml/kg);

Group III: Standard control (High fructosediet + Metformin 50 mg/kg)

Group IV: Hs.Cr 100 mg/kg (High fructosediet + Hs.Cr 100 mg/kg);

Group V: Hs.Cr 300 mg/kg (High fructosediet + Hs.Cr 300 mg/kg);

Group VI: Hs.Cr 500 mg/kg (High fructosediet + Hs.Cr 500 mg/kg).

Glucose-induced hyperglycemia

Administration of glucose (Merck, Germany)10% w/v in drinking water ad libitum on chronicbasis is found to induce hyperglycemic state (36).This method was adopted to evaluate the hypo-glycemic effects of Hs.Cr in Swiss albino mice ofboth sexes. Mice weighing 18-30 g were randomly


divided into different groups as mentioned below inthe experimental design. All the animals were given10% w/v glucose in drinking water for a period of28 days except the animals in the normal controlgroup that were given drinking water only. Animalswere intraperitoneally administered different dosesof either Hs.Cr or a standard drug according to theexperimental design. After four weeks of treatment,mice were fasted overnight (12 h) and their fastingblood glucose concentration was determined by glu-cometer from the tail vein.


Group II: Positive control (Normal saline 1mL/kg + 10% w/v glucose);

Group III: Standard control (Metformin 100mg/kg + 10% w/v glucose);

Group IV: Hs.Cr 100 mg/kg (Hs.Cr 100mg/kg + 10% w/v glucose);

Group V: Hs.Cr 300 mg/kg (Hs.Cr 300mg/kg + 10% w/v glucose);

Group VI: Hs.Cr 500 mg/kg (Hs.Cr 500mg/kg + 10% w/v glucose).

Collection of blood and serum and biochemical

analysis

After completion of the required study period,overnight fasted rats were anesthetized by intraperi-toneal injection of ketamine 50 mg/kg (Ketasol,Indus Pharma, Pakistan) and diazepam 5 mg/kg(Valium, Roche, Pakistan) to obtain blood by car-diac puncture. The blood was allowed to clot atroom temperature for 15 min and then centrifuged at4500 rpm for 15 min to obtain the serum in the cen-trifuge machine (Hettich Zentrifugen, Germany).Biochemical analysis for serum triglycerides (TG),total cholesterol (TC) and high density lipoproteins(HDL) was performed according to the protocol pro-vided with standard kits (Human Diagnostics,Wiesbaden, Germany). The results were obtained byrunning the respective samples, standards andreagent blanks, on Merck microlab 300 (MerckGermany). The serum low density lipoprotein(LDL) was estimated indirectly by usingFriedewald's formula (37) as given below:

LDL (mg/dL) = TC ñ HDL ñ TG / 5

Figure 1. Acute hypoglycemic effects of different doses of Hs.Cr in normoglycemic rats. (Values are expressed as the mean ± SEM and n= 6. Two way ANOVA is applied and p values are considered significant as p < 0.01 (**) and p < 0.001 (***). The extract treated groupsand standard control were compared to normal control)

Table 1. Phytochemical analysis of Heliotropium strigosum (Hs.Cr).

No. Phytochemical constituents Results

1. Alkaloids +

2. Saponins ++

3. Flavonoids -

4. Anthraquinones -

5. Tannins +

6. Glycosides -

7. Coumarins +

(+) sign indicates presence and (-) sign indicates the relative absence of the constituents in the crude extract ofHeliotropium strigosum (Hs.Cr).


Change in body weight

The body weight was measured on daily basisfor each animal. However, the difference in the bodyweight at the start of treatment (Wi) and at the end oftreatment (Wf) was used as a measure of change inthe body weight expressed in grams (g).


All the results were represented as the mean ±SEM. One way and two way ANOVA withBonferroni post hoc test was employed to demon-strate the level of significance i.e., p value < 0.05was considered significant. The positive controlswere compared to normal controls and all othergroups were compared to positive controls for sta-tistical significant difference between them. Thedata were compiled and statistically analyzed byusing GraphPad Prism version 5.00 for Windows,GraphPad Software (San Diego California, USA).

RESULTS

Preliminary phytochemical analysis and acute

toxicity assessment

The secondary plant metabolites such as alka-loids, saponins, tannins, and coumarins were detect-ed in the aqueous methanolic extract of H. strigosum(Hs.Cr) by preliminary phytochemical analysis assummarized in Table 1. The extract was safe untilthe dose of 3 g/kg in toxicity studies as no signs oflethality were observed.

Acute hypoglycemic effects in normoglycemic

rats

There was no significant difference in the fast-ing blood glucose (FBG) concentration in all thegroups at 0 h. The FBG concentration was signifi-cantly reduced in the standard group receivingglibenclamide 5 mg/kg, as well as in the groups treat-

Figure 2. Effects of chronic administration of different doses of Hs.Cr on hyperglycemia in alloxan-induced diabetic rats. (Values areexpressed as the mean ± SEM and n = 6. Two way ANOVA is applied and p values are considered significant as p < 0.001 (***). Positivecontrol was compared to normal control, while all extract treated groups and standard control were compared to positive control)

Table 2. Percentage glucose reduction by H. strigosum (Hs.Cr) in different animal models.

Groups Normoglycemic rats Alloxanized rats Fructose-fed rats Glucose-fed mice

Standard control 38% 67% 23% 42%

Hs.Cr 100 mg/kg 17% 36% 5% 26%

Hs.Cr 300 mg/kg 22% 56% 11% 36%

Hs.Cr 500 mg/kg 27% 62% 12% 41%

Normoglycemic Std. Control = Glibenclamide 5 mg/kg; Alloxan Std. Control = Glibenclamide 10 mg/kg; Fructose-fed Std. Control =Metformin 50 mg/kg; Glucose-fed mice Std. Control = Metformin 100 mg/kg.


ed with Hs.Cr at doses of 300 and 500 mg/kg at 1, 2,4 and 6 h after treatment, respectively (Fig. 1). Thesignificant reduction in FBG concentration by Hs.Cr100 mg/kg was evident only after 6 h of extractadministration as compared to normal controls. Thedecrease in FBG concentration was in a dose depend-ent manner (Fig. 1). The percentage reductions ofFBG concentration as compared to basal values indifferent groups are shown in Table 2.

Effects of H. strigosum in alloxan induced diabet-

ic rats

Effects of H. strigosum on fasting blood glucoseconcentration

The FBG concentration was significantlyraised in all the animals after 72 h of alloxan injec-tion as compared to normal control receiving normalsaline (data not shown). The FBG concentration inall the alloxanized rats remained elevated until day1 of the study as compared to normal control rats.Assessment at day 3 after onset of treatment showeda significant decrease in FBG concentration inglibenclamide 10 mg/kg and Hs.Cr 100, 300, and500 mg/kg treated groups as compared to positivecontrol (Fig. 2). FBG concentration in positive dia-betic control was significantly raised in comparison

to normal control (Fig. 2). A similar effect was seenon the 7th and 15th day of assessment of FBG con-centration with more pronounced decrease inglycemia with elapsing time as compared to diabet-ic controls (Fig. 2). The hypoglycemic effect of theHs.Cr 500 mg/kg was more pronounced on day 15and almost in the normal fasting glucose range (Fig.2). The percentage FBG concentration fall in differ-ent groups in comparison to the day 1 FBG concen-tration is shown in Table 2.

Effects of H. strigosum on total cholesterol, triglyc-erides, LDL and HDL

The total cholesterol and triglycerides concen-trations were significantly raised in positive controlrats as compared to normal rats (Fig. 3a and 3b). TheHs.Cr showed a significant dose-dependent decreasein the total cholesterol and triglyceride concentra-tion as compared to positive control. The Hs.Cr atdose of 500 mg/kg normalized the total cholesterollevels. The serum HDL concentration was signifi-cantly decreased in positive controls as compared tonormal controls (Fig. 3c). Interestingly, there was asignificant increase in serum HDL concentrationafter treatment with Hs.Cr at 300 and 500 mg/kg(Fig. 3c). The serum LDL concentration was

Figure 3. Effects of chronic administration of different doses of Hs.Cr on lipid profile in alloxan-induced diabetic rats. (Values areexpressed as the mean ± SEM and n = 6. One way ANOVA is applied and p values are considered significant as p < 0.01 (**), and p <0.001 (***). ns = not signifikant. Positive control was compared to normal control, while all extract treated groups and standard controlwere compared to positive control)


decreased in a dose-dependent manner with Hs.Cras compared to positive control, which in turn,showed significantly raised LDL concentration ascompared to normal control animals (Fig. 3d).

Effects of H. strigosum on body weightAlloxan induced diabetes causes a significant

weight loss due to muscle wasting. The loss ofweight is a typical clinical observation in type I dia-betes and was similarly observed in the alloxaninduced rat model of type I diabetes (Fig. 4). A neg-ative body weight change was observed in positivecontrols as compared to normal controls. The Hs.Crprevented the weight loss in a dose-dependent pat-tern, while highest weight gain was observed in

glibenclamide 10 mg/kg group (Fig. 4). These datashow that the effect of Hs.Cr on weight gain wasintermediate as compared to glibenclamide.

Effects of H. strigosum in high fructose-fed rats

Effects of H. strigosum on fasting blood glucoseconcentration

Fructose does not cause severe hyperglycemiaas evident with alloxan and thus, the slightly raisedblood glucose levels associated with fructose feed-ing are referred to as impaired fasting blood glucoselevels. At week 1 of assessment, the FBG concen-tration was significantly raised in positive controlrats as compared to normal rats, but only standardcontrol metformin 50 mg/kg (Glucophage, Merck,

Figure 4. Effects of chronic administration of different doses of Hs.Cr on change in body weight in alloxan-induced diabetic rats. (Valuesare expressed as the mean ± SEM and n = 6. One way ANOVA is applied and p values are considered significant as p < 0.001 (***).Positive control was compared to normal control, while all extract treated groups and standard control were compared to positive control)

Figure 5. Effects of chronic administration of different doses of Hs.Cr on FBG concentration in fructose-fed rats. (Values are expressed asthe mean ± SEM and n = 6. Two way ANOVA is applied and p values are considered significant as p < 0.01 (**), and p < 0.001 (***). ns= not significant. Positive control was compared to normal control, while all extract treated groups and standard control were compared topositive control)


Pakistan) depicted significant reduction (Fig. 5) inFBG concentration as compared to positive control.The Hs.Cr exerted no statistically significant hypo-glycemic effects at all administered doses. However,after week 2, the Hs.Cr at dose of 300 and 500mg/kg significantly decreased FBG concentration.The hypoglycemic action of Hs.Cr at the 100 mg/kgdose became significant after four weeks of treat-ment (Fig. 5). In addition, the Hs.Cr at doses 300and 500 mg/kg significantly prevented any increasein glycemia from week 3 of assessment until week 6of the study as compared to positive control group(Fig. 5). The percentage decrease in FBG concentra-tion by metformin and Hs.Cr at doses 100, 300, and500 mg/kg was 23, 5, 11 and 12%, respectively, ascompared to the diabetic control at the end of week6 (Table 2).

Effects of H. strigosum on total cholesterol, triglyc-erides, LDL and HDL

The serum total cholesterol concentration wassignificantly increased in fructose-fed positive con-trols as compared to normal controls (Fig. 6a). Theadministration of Hs.Cr at doses 300 and 500 mg/kgsignificantly protected against the hypercholes-

terolemic effects of fructose (Fig. 6a). The serumtriglyceride concentration was elevated in positivecontrols as compared to normal rats. The Hs.Cr atdose 100 mg/kg showed no significant reduction oftriglycerides, but Hs.Cr at doses 300 and 500 mg/kgwere more effective in preventing hypertriglyc-eridemia (Fig. 6b). The serum HDL levels were alsosignificantly impaired in fructose-fed rats as com-pared to normal controls (Fig. 6c). Only Hs.Cr atdoses 300 and 500 mg/kg significantly increasedHDL levels as compared to positive controls. Theeffect of Hs.Cr at dose 500 mg/kg was almost com-parable to the standard control metformin (50mg/kg) (Fig. 6c). The indirectly estimated LDL con-centration was significantly increased in positivecontrols as compared to normal controls. The Hs.Crat dose 100 mg/kg showed no significant decrease inLDL concentration, but the Hs.Cr at doses 300 and500 mg/kg showed a more pronounced lowering ofLDL concentration (Fig. 6d).

Effects of H. strigosum on OGTTThe blood glucose concentration was signifi-

cantly elevated in positive controls as compared tonormal controls at all time points of assessment. The

Figure 6. Effects of chronic administration of different doses of Hs.Cr on lipid profile in fructose-fed rats. (Values are expressed as themean ± SEM and n = 6. One way ANOVA is applied and p values are considered significant as p < 0.001 (***). ns = not significant.Positive control was compared to normal control, while all extract treated groups and standard control were compared to positive control)


Hs.Cr at doses 100, 300 and 500 mg/kg significant-ly improved the glucose tolerance at all time pointswhen compared to fructose-fed rats (Fig. 7a). Thearea under the glucose concentration time curve(AUC) was significantly larger in positive controlsas compared to normal rats. The administration ofmetformin (50 mg/kg) and of different doses ofHs.Cr significantly decreased the AUC as comparedto positive controls (Fig. 7b).

Effects of H. strigosum on glucose induced hyper-

glycemia

Chronic administration of glucose results inhyperglycemia in experimental animals (36). Mice

in the positive control group were receiving 10%w/v glucose in drinking water ad libitum andshowed significant development of hyperglycemiaafter 28 days as compared to normal control mice onwater only (Fig. 8). The Hs.Cr at doses 100, 300 and500 mg/kg significantly prevented hyperglycemia ascompared to positive control mice (Fig. 8), confirm-ing antihyperglycemic effect of H. strigosum. Thepercentage decrease in FBG concentration by stan-dard control and Hs.Cr different doses are shown inTable 2.

DISCUSSION

Diabetes mellitus is a heterogeneous diseasewith absolute to relative insulin deficiency associat-ed with insulin resistance. Chronic hyperglycemia isa major problem in diabetes mellitus (38). The pro-longed hyperglycemia leads to the synthesis ofadvanced glycation end products (AGEs) and, con-sequently, the development of various microvascu-lar complications (3) appearing later in the course ofthe disease (39). Interventions aimed at achievingbetter blood glucose level control with minimumside effects are urgently needed due to the increas-ing incidence of diabetes. In the present study, weevaluated the hypoglycemic and antihyperglycemiceffects of the aqueous methanolic extract of the aer-ial parts of the H. strigosum (Hs.Cr) in different ani-mal models of diabetes. The Hs.Cr effectivelydecreased FBG concentration in normoglycemic ratsat doses 300 and 500 mg/kg, while the effects atdose 100 mg/kg were slow in onset. However, thehypoglycemic effect of Hs.Cr was not as severe asseen with standard control glibenclamide 5 mg/kg(Fig. 1), which is well known for causing hypo-glycemic coma due to inhibition of compensatoryglucagon release (40, 41).

Figure 7. Effects of different doses of Hs.Cr on OGTT in fructose-fed rats. (Values are expressed as the mean ± SEM and n = 6. a. Twoway ANOVA and b. One way ANOVA is applied and p values are considered significant as p < 0.01 (**), and p < 0.001 (***). Positivecontrol was compared to normal control, while all extract treated groups and standard control were compared to positive control)

Figure 8. Effects of different doses of Hs.Cr on FBG concentrationin glucose (10%) fed mice. (Values are expressed as the mean ±SEM and n = 6. One way ANOVA is applied and p values are con-sidered significant as p < 0.001 (***). Positive control was com-pared to normal control, while all extract treated groups and stan-dard control were compared to positive control)


Alloxan is known to induce a hyperglycemicstate by destroying the pancreatic β cells via redoxmediated mechanisms (42). It has been well docu-mented that the FBG concentration is elevated inalloxan injected rats (26, 27), which was alsoobserved in our rat model. The Hs.Cr administrationin alloxan-induced diabetic rats prevented hyper-glycemia in a dose-dependent manner and theeffects of Hs.Cr 500 mg/kg were almost approach-ing the standard control glibenclamide 10 mg/kg(Fig. 2). Long standing hyperglycemia in diabetesleads to vascular complications, causing dysfunc-tion, damage and failure of various organs includingeyes, kidneys, nerves, heart and blood vessels (43).The auto-oxidation of glucose in hyperglycemicstate can lead to lipid peroxidation, altered antioxi-dant defence and further impairments in glucosemetabolism, which are mediated by reactive oxygenspecies (ROS) (44ñ46). The H. strigosum, throughits antioxidant (13) and antihyperglycemic effects,may protect against the oxidative stress in diabetes.The Hs.Cr hypoglycemic action possibly invovlesthe potentiation of the insulin effect (47).

The Hs.Cr exerted beneficial effects on thelipid profile by lowering TC, TG, and LDL concen-trations and increasing HDL concentration (Fig. 3).The hypocholesterolemic effects might be evidentdue to improved glycemic control along withdecreased cholesterol absorption or biosynthesis andmodifications in lipoprotein metabolism (48ñ50).The hypertriglyceridemia observed in diabetic rats ismost likely due to inability to activate the lipopro-tein lipase during insulin deficiency (51, 52). Thehypotriglyceridemic effect of Hs.Cr suggests that itmay be able to prevent against increased risk ofmyocardial infarction, stroke, coronary heart diseaseand atherosclerosis associated with diabetes-linkedhypertriglyceridemia (53ñ56). The dose-dependentdecrease in serum LDL is perhaps attributed to bet-ter glycemic control and/or may be due toinsulinotropic activity of the extract (57, 58). Thecoronary heart disease risk is increased with highcirculating LDL levels associated with low levels ofHDL (59). Hs.Cr at doses 300 and 500 mg/kg effec-tively raised HDL levels, which is probably due toincreased extra-hepatic removal and transport ofcholesterol to the liver for clearance (47, 60).Antidyslipidemic effects of the Hs.Cr may con-tribute to the reduction of the risk of cardiovascularcomplications associated with diabetes.

Insulin deficiency in alloxanized rats leads toincreased gluconeogenesis and glycogenolysis withwasting of muscle and loss of tissue proteins, result-ing in loss of body weight (61, 62). The Hs.Cr pre-

vented weight loss in a dose-dependent manner andthe Hs.Cr related weight gain was more favorablethan that seen with glibenclamide, which is know tocause excessive weight gain due to its insulin secre-tagogue action (63). Hs.Cr has been reported as lax-ative (6ñ8) but we did not observe any severe laxa-tive effect leading to excessive weight loss at leastwith the administered doses.

Metabolic syndrome and type II diabetesprevalence has increased dramatically in recentyears (64). Therefore, the effects of Hs.Cr on FBGand lipid profile were investigated in fructose-fedrats that resemble the metabolic syndrome andinsulin resistance state seen in early type II diabetes.High amounts of fructose in the diet cause insulinresistance manifested by deleterious metaboliceffects like hyperinsulinemia, hyperglycemia, glu-cose intolerance, hypertriglyceridemia and hyper-tension in rats. The fructose-fed rat model mimicsthe metabolic syndrome (syndrome X) in humans.Fructose-fed rats have shown impaired glucose oxi-dation in liver, skeletal muscles and adipose tissuedue to insulin stimulation (34, 65, 66). The fructosefeeding impaired FBG concentration in agreementwith previous findings (33, 67) and the administra-tion of Hs.Cr prevented hyperglycemia in a dose-dependent manner. The Hs.Cr anti-hyperglycemiceffect might be due to decreased glucose output bythe liver and/or increased peripheral glucose utiliza-tion due to improved insulin sensitivity such as seenwith standard control metformin (67). The Hs.Cralso improved the glucose tolerance in a dose-dependent manner in OGTT, which was conductedin the last week of fructose feedings. These findingsdemonstrate that the Hs.Cr possibly improvesinsulin resistance and prevents impaired glucose tol-erance, which are common features of syndrome Xand early risk factors for developing type II diabetes(68), thereby increasing the risk of cardiovasculardisease (69).

Fructose is more lipogenic and leads to hyper-cholesterolemia and hypertriglyceridemia (70).Increased levels of serum TG contribute to insulinresistance (71, 72). The Hs.Cr has shown a dose-dependent hypocholesterolemic and hypotriglyceri-demic effects. The increase in serum HDL and thedecrease in serum LDL were only observed at doses300 and 500 mg/kg of the Hs.Cr. These effects maystem from the ability of the extract to reduce theinsulin resistance.

High plasma glucose levels as observed in dia-betes, are deleterious to the pancreatic β cells, and areoften reported to cause glucose toxicity or glucosedesensitization. Lowering of glucose concentration is


associated with increase in insulin sensitivity andinsulin release (73). Administration of the Hs.Cr toglucose fed mice protected against glucose toxicityby lowering the FBG concentration. Our resultsdemonstrated that H. strigosum was effective in con-trolling blood glucose and correcting dyslipidemia indiabetes mellitus, however, the contents of the extractexerting these effects still need further investigation.

CONCLUSION

The aqueous methanolic extract of H. strigo-sum (Hs.Cr) was effective both as a hypoglycemicand anti-hyperglycemic agent in the experimentalmodels of type I and type II diabetes. The extractalso corrected the dyslipidemia associated withalloxan-induced diabetes and fructose feeding.Thus, further studies aimed at isolating the activeagents in aqueous methanolic extract of H. strigo-sum and exploring the underlying mechanisms ofaction are highly encouraged.

Acknowledgments

We are indebted to Prof. Dr. Mahmood Ahmad,Dean, Faculty of Pharmacy and Prof. Dr. NaveedAkhtar, Chairman, Faculty of Pharmacy, for theirkind support and for facilitating this research project.We are thankful to Mr. Abdul Hameed for his help inplant selection and identification. We are also thank-ful to Ms. Joyce Zaftis for proof reading the article.

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Received: 9. 11. 2015


Marine natural products represent a potentiallyrich and largely untapped source of novel bioactivechemical entities (1). The marine natural productsare particularly interesting because many of themhave unique structures, and may thus exhibit unusu-al properties (2). Marine organisms account formore than half of the worldís total biodiversity andthe marine-derived natural products serve a varietyof purposes that are not fully understood. Some have

medicinally interesting properties and are reportedto be active against various diseases, including can-cer (3).

Sarcophyton is one of the most widely distrib-uted soft coral genera in the tropical and sub-tropi-cal oceans (4). To date, around 30 species from thisgenus have been sampled and tested for bioactivesecondary metabolites. Extracts from the speciesexhibited a range of interesting pharmacological

CYTOTOXIC EFFECTS OF THE RED SEA SOFT CORAL SARCOPHYTONTROCHELIOPHORUM

HESHAM R. El-SEEDI1,2,3*, MOHAMED GOMAA4, MOUSA MAALI SALEM4, KHALED BENCHOULA5, HAGER M. KESHK3, NERMEEN YOSRI3, AHMED AYESH4,

AHMED M. ASKER3, KAWTHER SOLIMAN4, ZEINAB HAMZA6, HAGER M. MANSOUR3, AHMED ELKHATEEB7 and SHADEN A. M. KHALIFA8,9

1Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia2Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre,

Box 574, SE-751 23, Uppsala, Sweden3Department of Chemistry, Faculty of Science, El-Menoufia University, 32512 Shebin El-Kom, Egypt

4Biology Department, Faculty of Sciences and Arts, University of Jeddah, Khulais, Kingdom of Saudi Arabia

5Department of Pharmaceutical Chemistry, Faculty of Pharmacy, International Islamic University Malaysia,Kuantan 25200, Pahang, Malaysia

6Marine Toxins Laboratory, Food Toxins and Contaminants Department, National Research Centre, Dokki,Cairo, Egypt

7Pharmaceutical and Drug Industries Research Division, Phytochemistry and Plant Systematic Department,National Research Centre, Egypt

8Department of Experimental Hematology, Karolinska University Hospital, SE-141 86 Stockholm, Sweden

9Department of Molecular Biosciences, Stockholm University, the Wenner-Gren Institute, SE-106 91 Stockholm, Sweden

Abstract: The present study describes the in vitro cytotoxic effects of soft coral (Sarcophyton trocheliophorum).Soft corals of genus Sarcophyton were reported to contain compounds that are active against brine shrimp andpromote paclitaxel cytotoxicity in the human colon cancer Caco-2 cell line. The n-hexane extract of the softcoral Sarcophyton trocheliophorum induced significant dose-dependent toxicity (LC50 96.7 ppm) compared withethyl acetate (LC50 120 ppm). We reported the most active cytotoxic level to be correspondence to LC50 valuesof 20.2, 59.2 ppm and 18.9 and 26 ppm. Accordingly, bio-assay guided fractionation was conducted to identi-fy the bioactive compounds. Arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid were charac-terized based on GC-MS analyses. Our results demonstrate the value of marine products as a natural source ofmedicinally interesting cytotoxic compounds.

Keywords: cytotoxic activity, GC-MS, Sarcophyton trocheliophorum, soft coral, unsaturated fatty acid

1587

* Corresponding author: E-mail: [email protected]; [email protected]; address: Division of Pharmacognosy, Department ofMedicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23, Uppsala, Sweden; phone: +46-18-4714207. fax: +46-18-509101

1588 HESHAM R. El-SEEDI et al.

properties, including HIV inhibition (5), neuropro-tection (6), antifeedant, antifouling and anti-inflam-matory activities (7, 8).

The current study aimed at investigating themarine chemical and cytotoxic properties exhibitedby the bioactive compounds extracted fromEgyptian soft coral S. trocheliophorum that has awide range of potential biological properties.Bioassay-guided fractionation of trocheliophorumspecies would facilitate the isolation and identifica-tion of the compounds responsible for their activity,which could then be evaluated as potential drug can-didates or lead compounds. Recently, we justifiedthe antibacterial effect of the red sea soft coralSarcophyton trocheliophorum and reported the iden-tification of 5S-3-[(3E,5S)-5-hydroxy-3-hepten-6-yn-1-yl]-5-methyl-2(5H)-furanone (9), as part of ourongoing investigations on Egyptian soft corals withparticular emphasis on the isolation and structureelucidation of potential bioactive compounds.

EXPERIMENTAL

Materials and methods

Chemistry Accelerating gradient chromatography (AGC)

was performed using variable-length MPLC glasscolumns (Baeckstrˆm Separo AB, Lidingˆ,Sweden) with inner diameters of 4, 2.5, and 1.5 cm,packed with silica gel 60, 40-63 µm (Merck), and anFMI Lab pump, model QD (Fluid Metering Inc.,Oyster Bay, NY), delivering a flow rate of 15-18mL/min. Fractions of 20 mL were collected manual-ly (10). Analytes were eluted from the columns bycontinuous gradient elution running from hexane,through CH2Cl2, EtOAc, and MeOH, to H2O gener-ated by a Separo constant volume mixing chambercombined with an open reservoir. The mixing cham-ber initially contained 100 mL of hexane and thefirst 15-20 premixed binary gradient mixtures ofgradually increasing polarity collected in the reser-voir, then each 15-30 mL were successively fed tothe reservoir during the separation.

TLC was performed on pre-coated aluminumsheets [silica 60 F254, 0.25 mm (Merck, Darmstadt,Germany)] and preparative TLC on pre-coated glasssheets [silica 60 F254, 0.5 mm (Merck)], with UVlight detection (254 and 366 nm) followed by spray-ing with vanillin-sulfuric acid reagent and heating at120OC.

Gas chromatography-mass spectrometry (GC-MS) was done using a Varian 3400 GC connected toa Finnigan SSQ 7000 quadrupole mass spectrome-ter, (Sweden) equipped with a split/split less injector

(split less mode, 30 s; injector temperature, 230OC;carrier gas - helium with a constant pressure of 10psi). A DB-WAX capillary column (30 m, 0.25 mmID, and 25 µm film thickness, J & W USA) wasused. The temperature program was: 40OC for 1 minincreased with 4OC/min up till 235OC and hold at235OC for 10 min. Transfer line connecting GC tothe MS was isothermally set to 235OC throughoutthe analysis. Filament off time was 4 min and theMS ion source temperature 150OC; mass spectrawere obtained at 70 eV with a mass range of 30-400m/z. Mass spectra of compounds were compared tothe Finnigan NIST-2005 (National Institute ofStandard and Technology) MS library and the avail-able reference compounds (11).

Soft coral material

Soft coral (S. trocheliophorum) colonies werecollected by trained divers working in the harborareas of Hurghada on the Red Sea in Egypt, withinan area ranging from 27O00 to 27O45 N and from33O30 to 34O00 E. The soft coral Sarcophyton troche-liophorum (common name: toadstool coral or leathercoral, family Alcyoniidae) is native of the Red Seareefs. The samples were identified and documentedby Hurghada Marine Station of the NationalInstitute of Oceanography and Fisheries.

Extraction and isolationThe collected samples were immediately trans-

ported to the laboratories of the Hurghada MarineStation, National Institute of Oceanography andFisheries. Samples were washed twice with filteredseawater then briefly, with distilled water. Thewashed samples were blended with absolute ethanol(1 : 1, w/v) and centrifuged (2.57 kg for 10 min) thenextracted with n-hexane at room temperature threetimes with occasional stirring and filtered to give265.1 g of a hexane extract.

The macerate was then extracted three timeswith MeOH for 7 days each to give 311.6 g of aMeOH extract. The extract was partitioned betweenEtOAc and H2O to give 165.6 g and 121.2 g afterfreeze-drying. An interfacial residue, 24.8 g wasalso produced. The n-hexane extracts (60 g) wereadsorbed onto silica gel (120 g) and chro-matographed on a silica gel (280 g) column elutedwith continuous hexane-CH2Cl2, CH2Cl2 to MeOHgradients. The eluted tubes from AGC columnswere evaluated by TLC and resulted in 7 main frac-tions (H1-H7). The most active fractions (H2, H3)were subjected to further purification over repeatedAGC elution with petrolium ether-CHCl3 (1 : 1, v/v).Three compounds: arachidonic (1), eicosapen-

Cytotoxic effects of the Red Sea soft coral Sacrophyton trochliophorum 1589

taenoic (2) and docosahexaenoic acids (3) wereidentified from S3H2 sub-fraction (Fig. 1). Theacids were then methylated by heating with 5%anhydrous HCl in MeOH and the fatty acids methylesters were further analyzed by GC-MS (12).

Methyl arachidonate 1 [(5E,8E,11E,14E)-methyleicosa-5,8,11,14-tetraenoate]; M.F. C21H34O2 at m/z318 with fragmentation patterns; 150 [M - 168], 119[M - 199], 105 [M - 213], 91 [M - 227], 79 [M -239], 67 [M - 251], 55 [M - 263], 41 [M - 277] and29 [M - 168].

Methyl eicosapentaenate 2 [(2E,4E,6E,8E,10E)-methyl eicosa-2,4,6,8,10-pentaenoate]; M.F.C21H32O2 at m/z 316 with fragmentation patterns;245 [M - 71], 201 [M - 115], 143 [M - 173], 131 [M- 185], 117 [M - 199], 105 [M - 211], 91 [M - 225],79 [M - 237], 67 [M - 249] and 55 [M - 261].

Methyl docosahexaenate 3 [(4E,7E,10E,13E,16E,19)-methyl docosa-2,4,6,8,10-hexaenoate]; M.F.

C23H34O2 at m/z 342 with fragmentation patterns;313 [M - 29], 311 [M - 31], 119 [M - 223], 108 [M- 234], 105 [M - 237], 91 [M - 251], 67 [M - 275],79 [M - 263], 55 [M - 287] and 41 [M - 301].

Biological screening

Cytotoxic activity (brine shrimp lethality bioas-

say)

The cytotoxicity of the fractions was assessedusing the brine shrimp lethality bioassay (13).Briefly, 1 g of brine shrimp (A. salina) eggs wasallowed to hatch in artificial brine (prepared by dis-solving 36 g of instant ocean salt in 1 L of distilledwater). The hatching was induced at room tempera-ture, in a solution with a constant supply of oxygen,maintained at a pH between 7 and 8. The phototrop-ic nauplii were collected 24 h after hatching and 10shrimps were transferred to each sample vial using aPasteur pipette.

Figure 1. Structures of the fatty acids as identified from the H2 fraction

Figure 2. The brine shrimp toxicity curve of the hexane fractions (H1-H7). LSD at p ≤ 0.05 = 4.9505

Docosahexaenoic (3) acids


The extracts under investigation were exam-ined with the bio-assay in triplicate at initial con-centrations of 25, 50, 100, 250, 500, and 1000 ppmin vials containing 5 mL of brine and 10 shrimp. Thenumber of surviving shrimp after 24 h was reportedand the estimation of the mortality percentage,which serves as a measure of toxicity level, was con-ducted (14). The median lethal concentration (LC50,the concentration that resulted in 50% mortality)was determined by fitting the corresponding dose-response curve to a sigmoidal non-linear regressionmodel. All reported LC50 values represent meansfrom three experimental runs.


The present study was performed with particu-lar emphasis on characterization of cytotoxic profileof S. trocheliophorum extract, fractions and sub-fractions using brine shrimp lethality assay. Thecytotoxicity of n-hexane and EtOAc extracts were

initially tested at different concentrations (25, 50,100, 250, 500 and 1000 ppm). The n-hexane extractexhibited significant dose-dependent toxicity at con-centrations of 50 ppm and above, causing 100%mortality at concentrations of 500 and 1000 ppm.The n-hexane extract induced a slightly higher mor-tality than EtOAc at all investigated concentrationswith an LC50 of 96.7 ppm.

To find out the bioactive compounds responsi-ble for the potent activity, the sub-fractions weresubjected to further characterization and three fattyacids (Fig. 1) were successfully isolated andexposed to methylation, leading to the formation ofmethyl arachidonate 1, methyl eicosapentaenate 2

and methyl docosahexaenate 3, then consequentlyidentified by using GC-MS analysis.

Fractionation of the most active crude extractwas performed and yielded seven fractions (H1-H7). H2 fraction induced the most prominent effectfollowed by H3 with an LC50 value of 20.2, 59.2ppm, respectively, whereas H4-H7 fractions dis-

Figure 3. Cytotoxicity curve of the H2 sub-fractions (S1H2-S5H2). LSD at p ≤ 0.05 = 3.3949

Figure 4. The brine shrimp toxicity curve of H3 sub-fractions (S1H3-S5H3). LSD at p ≤ 0.05 = 3.3949

Cytotoxic effects of the Red Sea soft coral Sacrophyton trochliophorum 1591

played no activity as illustrated in Table 1. Both H2and H3 fractions induced complete mortality atconcentrations of 250 ppm and above. Furthermore,H2 fraction showed significant mortality at 25 ppmwhereas the critical threshold for H3 was at 50 ppm(Fig. 2).

Further evaluation of the hexane sub-fractionsexhibited significant cytotoxicity activity of S3H2and S3H3. S3H2 and S3H3 showed cytotoxiceffects against brine shrimp with a LC50 value of18.9, 26.0 ppm, respectively, as illustrated inFigures 3, 4 and Table 1. The maximum effects werenoticed at 25 ppm, the increase of the LC50 value to100 ppm attributed to a complete mortality.

Several previous studies have indicated thatsoft corals of the genus Sarcophyton contain com-pounds that are active against brine shrimp (15),which are in most cases correlated reasonably wellwith cytotoxic and antitumor teratology properties(16). Many researchers use the brine shrimp test asa preliminary screen for cytotoxicity due to the pos-itive correlation between brine shrimp lethality and9 KB (human nasopharyngeal carcinoma) cytotoxi-city (p = 0.036 and κ = 0.56). The median effectivedose (ED50) for a given compound or extract in thecytotoxicity assay is generally around one-tenth ofits median lethal concentration (LC50) in the brineshrimp test (17). The concentrations of the activesub-fractions (100 ppm) required to achieve 100%mortality in this work were consistent with the liter-ature (18). Moreover, the active compounds showedpotent and selective cytotoxic actions on the humancolon cancer Caco-2 cell line (19, 20).

Marine products provide a basis for the isola-tion of unique and potent bioactive compounds.Marine products are considered to be a potentialsource of new anti-cancer compounds; however,little is known about their chemical and pharma-cological potential. Anticancer activity has beenshown to be associated with a variety of polyun-saturated fatty acids (PUFAs) classes, such aseicosapentaenoic acid (EPA), docosahexaenoicacid (DHA) and arachidonic acid (12, 21). PUFAsare fatty acids with more than 2 double bonds,have been implicated in inhibiting the cancerdevelopment and the majority of marine-basedlong-chain polyunsaturated fatty acids(LCPUFAs) have showed a wide range ofchemotherapeutic efficacy (22). PUFAs integratewithin the cellular membranes thus sensitize thetumor cells to ROS-inducing cells and induce anti-cancer effect both in vitro and in vivo. In thisstudy, a number of polyunsaturated fatty acidshave been isolated from soft corals, namelyarachidonic acid, eicosapentaenoic acid anddocosahexaenoic acid that were attributed to thereported cytotoxic activity. The produced cytotox-icity was both time- and concentration-dependent.The high yield of the fatty acids and their closeeffect against human colon cancer Caco-2 cell lineindicate that the cytotoxicity is due to the presenceof PUFA rather than the fatty acid metabolism perse. In conclusion, we highlight the significantcytotoxic activity of marine polyunsaturated fattyacids including those occurring in the soft corals.This information helps to establish efficacioussources for modern treatment modalities to combatcancer (23).

CONCLUSION

Our study proved that fractions from hexaneextract of corals belonging to the genus Sarcophytonare highly active against the brine shrimp. Bio-assayguided fractionation strategy leads to isolation ofthree fatty acids with potent lethality effects. Thepurification and isolation of the marine active com-pounds provide a feasible platform for the futurepharmaceutical applications.

Acknowledgment

We are very grateful for financial support byHigh Impact Research Grant UM ñ MOHE:UM.C/625/1/HIR/MOHE//SC/09 from TheMinistry of Higher Education, Malaysia.

Table 1. LC50 values of the samples and the fractions indicating thehighest to lowest toxic effect.

Sample LC50 (ppm)

S3H2 18.9 ± 2.2

H2 20.0 ± 1.2

S3H3 26.0 ± 0.3

S2H3 56.0 ± 1.8

H3 59.2 ± 3.6

S2H2 102.6

H1 221.2

H4 384.8

H7 453.8

H6 473.1

H5 490.2

Hexane extract 96.7 ± 3.9

EtOAc extract 120 ± 4.3


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Received: 10. 11. 2015


With over 850 species, Ficus, of the plant fam-ily Moraceae, is one of the oldest, most successful,but understudied genera in modern pharmacognosyand has extensive distribution of secondary metabo-lites such as triterpenoids, phenolics, flavonoids,alkaloids, coumarins and sterols (1-4). Ficus, withintheir vasculatures, possess and secrete a latex-likematerial, affording protection from harm and pro-viding self-healing from physical injuries (5). Formany centuries, animals and humans have dependedon its fruit as a source of nourishment while otherplant parts such as the leaves, bark, and roots hasbeen utilized for medicinal purposes. The latex fromFicus species have been investigated for their anti-cancer activity as far back as the 1940ís (5) andextracts have demonstrated enhanced intracellularaccumulation of daunomycin in K562/R7 leukemiccells as well as cytotoxic effects on the growth ofmulti-drug resistant human sarcoma MES-SA/Dx5cells (5). Thus, the extracts play an adjunctive rolein multiple cancer prevention and cancer chemother-apy (5, 6). The antioxidant potential of the plant hasalso been reported where the extracts have been

shown to decrease lipid peroxidation and increaseantioxidant enzymes (7). Although the medicinalbenefits of Ficus in humans are based on historicaland anecdotal reports, with few modern clinical tri-als, ethno-medicinal uses suggest anti-neoplasticand anti-inflammatory actions (5).

Ficus burtt-davyi, known as Uluzi by the Zulupeople in KwaZulu-Natal, is highly adaptable to awide variety of habitats and has even been known togrow on larger trees (epiphytic) as a strangler fig, aswell as on rocks (epilithic) where the roots are ableto split the rocks in their search for nutrients (8). Thefruits of the plant are edible and also used as a laxa-tive by the locals in the Eastern Cape (8). To date,no information has been reported on the chemicalcomposition and antioxidant activity of this plant. Inthis study, we report on the isolation and identifica-tion of compounds from the leaves and stem bark ofF. burtt-davyi. In addition, we report on the antioxi-dant activity of the methanol (MeOH) extracts of theleaves, stem bark and selected isolated compoundsusing a multi-method approach due to the complex-ity of the mechanisms of antioxidant activity (9).

CHEMICAL CONSTITUENTS AND IN VITRO ANTIOXIDANT ACTIVITY OFCRUDE EXTRACTS AND COMPOUNDS FROM LEAVES AND STEM BARK

OF FICUS BURTT-DAVYI

OLUMUYIWA O. OGUNLAJA1, ROSHILA MOODLEY1, HIMANSU BAIJNATH2

and SREEKANTHA B. JONNALAGADDA1,*

1School of Chemistry and Physics, 2School of Life Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa

Abstract: Ficus burtt-davyi, (Moraceae), is an endemic species of South Africa. In this study, a phytochemicalanalysis of the leaves and stem bark of F. burtt-davyi resulted in the isolation of five triterpenes (lupeol, lupeolacetate, β-sitosterol, stigmasterol and campesterol), one carotenoid (lutein), a pheophytin (phytyl-7-ethyl-25-(methoxycarbonyl)-3,8,13,17-tetramethyl-26-oxo-12-vinyl-17,18-dihydro-2,20-ethanoporphyrin-18-propano-ate or pheophytin a) and one flavonoid (+)-catechin). The in vitro antioxidant study of the methanol extracts ofleaves and stem bark, (+)-catechin and pheophytin a using the 1,1-diphenyl-1-picrylhydrazyl (DPPH) free rad-ical scavenging assay, ferric reducing antioxidant power (FRAP) assay and hydrogen peroxide (H2O2) assayshowed significantly higher (p < 0.05) antioxidant activity for the methanol extract of the stem bark than theleaves, with IC50 values (in µg/mL) of 58.28 ± 5.05 for DPPH, 46.09 ± 0.06 for FRAP and 151.03 ± 1.60 µg/mLfor H2O2. The results suggest that the plant can be used as a therapeutic agent in alternative medicine for oxida-tive stress related degenerative diseases.

Keywords: pheophytin, triterpenes, flavonoids, antioxidants

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1594 OLUMUYIWA O. OGUNLAJA et al.

EXPERIMENTAL

General experimental procedures

The 1H, 13C and 2D-NMR spectra were record-ed on a Bruker AvanceIII 400 MHz spectrometer at400.22 MHz for 1H and 100.63 MHz for 13C.Chemical shifts (δ) are reported in ppm and cou-pling constants (J) in Hz. The 1H and 13C chemicalshifts of the deuterated chloroform (CDCl3) were7.24 and 77.0, respectively, referenced to the inter-nal standard, tetramethylsilane (TMS). Infrared (IR)spectra were recorded using a Perkin-ElmerUniversal ATR spectrometer. UV spectra wereobtained on a Hewlett Packard UV-3600 spec-trophotometer. Column chromatography (CC) wasperformed with Merck silica gel 60, (0.040ñ0.063mm). Thin layer chromatography (TLC) was per-formed on Merck 20 ◊ 20 cm silica gel 60, F254 alu-minum sheets. The spots were analyzed under UV(254 nm and 366 nm), visualized using 10% H2SO4

in MeOH followed by heating. Solvents (analyticalgrade) and other chemicals used were supplied byeither Merck (Darmstadt, Germany) or Sigma (St.Louis, USA) chemical companies.

Plant material

The leaves and bark from F. burtt-davyi werecollected on the Westville Campus of the Universityof KwaZulu-Natal (UKZN), Durban, South Africa,in June 2013. The plant was authenticated by Prof.Himansu Baijnath and a voucher specimen(Ogunlaja, O1) has been deposited in the Herbariumof the School of Life Sciences, UKZN, Westville.The stem bark and leaves of the plant were dried andground using a mini-industrial grinder (Wiley Mill).

Extraction, fractionation and isolation

The air-dried, powdered leaves (970 g) of F.burtt-davyi were sequentially exhaustively extractedwith (4 L ◊ 2) of n-hexane, dichloromethane(DCM), ethyl acetate (EtOAc) and MeOH for 24 husing an orbital shaker. The extracts were concen-trated by evaporation under vacuum at controlledtemperatures to obtain crude extracts of n-hexane(24.22 g), DCM (15.64 g), EtOAc (7.76 g) andMeOH (52.98 g). The n-hexane and DCM crudeextracts of leaves were combined due to similarTLC profiles (similar Rf values). This combinedextract (38.00 g) was subjected to column chro-matography (CC) using a gradient elution system ofn-hexane : EtOAc starting with 100% n-hexane until100% EtOAc was reached through 10% incrementsof EtOAc, collecting 100 mL fractions at each stage.Six main fractions (I-VI) were obtained and fraction

II yielded compound 1 (105 mg), which was elutedwith n-hexane: EtOAc (8 : 2, v/v). Fraction III (1.23g) was purified further to give compound 2 (18.6mg) and compound 3 (20.1 mg), respectively. TheEtOAc extract of the leaves (7.0 g) was separatedusing CC in a similar manner to yield seven majorfractions (I-VII) based on similar TLC profiles.Fraction II and III afforded compound 4 (30.5 mg)and compound 5 (10.5 mg), respectively.

The n-hexane and DCM crude extracts of thestem bark were combined due to similar TLC pro-files (similar Rf values). This extract (24.35 g) wassubjected to CC in a similar manner to that of theleaves to yield nine major fractions based on TLCprofiles. Fractions II and III gave compound 6 (4.5g) and compound 7 (4 g), respectively, which wereeluted with 100% DCM and recrystallized inMeOH. The MeOH extract from the stem bark waspartitioned with EtOAc and the EtOAc fraction wasconcentrated to yield 4 g of extract. This was sepa-rated on a 1.5 cm diameter column using CC and agradient of n-hexane: EtOAc (8 : 2, v/v) to 100%EtOAc (10% increments of EtOAc, 20 mL frac-tions) to afford compound 8 (23 mg).

Phenolic content and in vitro antioxidant assays

Estimation of total phenolic content (TPC)The total polyphenol content (TPC) of the

extracts from F. burtt-davyi was determined as gallicacid equivalent (GAE) according to the methoddescribed by McDonald et al. (10) with slight mod-ifications. Briefly, 200 µL of the extract (240µg/mL) was incubated with 1 mL of ten-fold dilutedFolin-Ciocalteau reagent and 800 µL of 0.7 MNa2CO3 for 30 min at room temperature.Absorbance values were determined at 765 nm on aShimadzu UV mini 1240 spectrophotometer(Shimadzu Corporation, Kyoto, Japan). All meas-urements were done in triplicate.

DPPH free radical-scavenging activity assay The capacity to scavenge the ëëstableíí free rad-

ical 1,1-diphenyl-1-picrylhydrazyl (DPPH) wasmonitored according to the procedure described byAhmad et al. (11) with some modifications. Variousconcentrations (7.5-500 µg/mL) of extracts and iso-lated compounds (3000 µL) made from a stock solu-tion (10 µg/mL) were mixed with 2850 µL MeOHsolution containing DPPH radicals. The mixture wasthen vortexed, and incubated for 30 min at roomtemperature. The scavenging activity was evidencedby a change in color from purple to yellow, whichwas further measured by the decrease in absorbanceat 517 nm using a Shimadzu UVñVis spectropho-

Chemical constituents and in vitro antioxidant activity of crude extracts... 1595

tometer. Ascorbic acid was used as the standardwhile MeOH served as the blank. The assays weredone in triplicate. The difference in absorbancebetween a test sample and the control (DPPH +MeOH) was expressed as percentage inhibition.

% inhibition = [(Ao - Asample / Ao) ◊ 100]where, Ao (control) = Absorbance of DPPH +methanol.

The IC50 value which is the inhibitory concen-tration in µg/mL of samples, or standard, necessaryto reduce the initial DPPH by 50% as compared tothe negative control was determined graphically byplotting the absorbance of DPPH as a function ofsample concentration in µg/mL for the standard andsamples.

Ferric reducing antioxidant power (FRAP) assayThe reducing power of the extracts and com-

pounds were determined according to the method ofOyaizu (1986) as described by Behera et al. (12)with some modifications. Various concentrations(7.5-500) µg/mL were mixed with 2.5 mL of sodiumphosphate buffer (0.2 M, pH 6.6) and 2.5 mL of0.1% potassium ferricyanide and the mixture wasincubated at 50OC for 30 min. After the addition of2.5 mL of 10% TCA, the mixture was centrifuged at3000 rpm for 10 min. The upper layer (2.5 mL) wasmixed with 2.5 mL of distilled water and 0.5 mL of0.1% ferric chloride, and the absorbance was meas-ured at 700 nm. The methanolic extract withoutreagents was used as a negative control while ascor-bic acid with the same concentrations was used aspositive controls.

Hydrogen peroxide-scavenging activity assayThe ability of the extracts to scavenge hydrogen

peroxide was determined according to the method ofRuch et al. (13). A solution of hydrogen peroxide (2mM) was prepared in 50 mM phosphate buffer (pH7.4). Samples of various concentrations (7.5-500µg/mL) were transferred into the test tubes, and theirvolumes were made up to 0.4 mL with 50 mM phos-phate buffer (pH 7.4). After addition of 0.6 mLhydrogen peroxide solution, tubes were vortexedand absorbance of the hydrogen peroxide at 230 nmwas determined after 10 min, against a blank.Phosphate buffer (50 mM) without hydrogen perox-ide was used as a blank and ascorbic acid was usedas the positive control. Hydrogen peroxide scaveng-ing ability was calculated using the following equa-tion:

Hydrogen peroxide scavenging activity = (1 -absorbance of sample/absorbance of control) ◊100

Statistical analyses

The experimental results were expressed as themean ± standard deviation (SD) of three replicatesand IC50 values were calculated by linear regression.The data were subjected to one way analysis of vari-ance (ANOVA) to determine significant differencesbetween means (p < 0.05) Tukey`s test was used forpost-hoc analyses. All the statistical tests were per-formed using Graphpad prism 6.0.


Structure elucidation of compounds from F. burtt-davyi

Compound 1, 2 and 3 were identified by usingspectral data (IR, UV, GC-MS, 1H-NMR, 13C-NMRand 2D NMR) and by comparison of the NMR datawith those reported in the literature. Compound 1

was identified as sitosterol (14), compound 2 ascampesterol (15) and compound 3 as stigmasterol(14).

Compound 4 was isolated as a dark green amor-phous solid. The mass spectrum of compound 4

obtained by HR-ESI-MS (positive mode) gave m/zvalue at 893.5530 corresponding to the molecular for-mula (C55H74N4O5) [M + Na] +, (calc. C55H74N4O5 Na894.1999). The IR spectrum of compound 4 showedabsorption bands at 2922, 3388, 1618 and 1376 cm-

1 corresponding to C(sp3), NH, CH=CH (vinylgroup) and CN, respectively. The UV spectrum ofcompound 4 showed absorption bands at 414 and669 nm. The data are consistent with the presence ofa porphyrin nucleus. The 1H-NMR spectrum of com-pound 4 showed characteristic peaks for chlorophyllderivatives. The 1H-NMR spectrum showed anupfield shift at δH -1.70 ppm (the NH proton fromthe pyrrole ring), a resonance at δH 6.1ñ7.9 ppm(vinyl group), meso-proton signals downfield at δH

9.47 (H-10), 9.33 (H-5) and δH 8.53 (H-20) ppm andδH 6.28 (dd, J = 1.6, 17.5 Hz, H-3a), δH 6.16 (dd, J= 1.5, 11.55 Hz, H-3b) ppm for a mono-substitutedvinyl group corresponding to the structure of pheo-phytin a. The characteristic phytyl side chain reso-nances at δH 1.60 (CH), 1.25 (CH2) and 0.97 (CH3)ppm were also observed. The 13C-NMR, DEPT 90and DEPT 135 spectra resolved fifty five carbon res-onances corresponding to eleven methyl, fourteenmethylene, eleven methine and twenty-one quater-nary carbons signals. These chemical shifts were inaccordance with those reported in the literature (16),thus, compound 4 was established as phytyl-7-ethyl-25-(methoxycarbonyl)-3,8,13,17-tetramethyl-26-oxo-12-vinyl-17,18-dihydro-2,20-ethanoporphyrin-18-propanoate (pheophytin a).


Compound 5 was obtained as a yellow coloredcompound and GC-MS data showed molecular ionpeak [M+] at m/z 568.9 which corresponds to themolecular formula C40H56O2. The UV spectrum ofcompound 5 showed absorption maxima at 454, 480and 430 nm. The 1H-NMR spectrum showed reso-nance in the olefinic region between δH 5.09-6.62ppm, methylene resonances of cyclohexene between

δH 1.33-1.44 ppm with a strong OH resonance at δH

4.23 ppm. Based on spectral data and those reportedin the literature, compound 5 was identified aslutein.

Compound 6 was isolated as a white solid. TheIR spectrum exhibited characteristic absorption fre-quencies at 3326, 878 and 1637 cm-1 typical of the O-H, unsaturated out of plane C-H and C=C bond

Figure 1. Chemical structures of compounds 1-8 isolated from F. burtt-davyi


vibrations, respectively. The absorptions bandsobserved at 1379 and 1452 cm-1 were due to the dis-tortion vibrations of CH3 groups and methylenicvibrations, respectively. The mass spectrum of com-pound 6 obtained by HR-ESI-MS (positive mode)gave m/z value at 449.3756 corresponding to themolecular formula (C30H50O) [M + Na] +, (calc.C30H50O Na 449.7174). The 1H-NMR spectrum forcompound 6 revealed the presence of seven tertiarymethyl singlets protons at δH 0.73, 0.76, 0.80, 0.92,0.94, 1.00 and 1.65 ppm; a multiplet at δH 3.18 ppm(H-3), and characteristic olefinic protons at δH 4.66(H-29 a) and 4.54 (H-29 b) ppm and a sextet at δH

2.39 ppm (19β -H). The DEPT 90 and 135 experi-ments resolved ten methylene, five methine and fivequaternary carbons. Based on spectral informationand those reported in the literature, compound 6 wasidentified as lupeol. The presence of lupeol in F.burtt-davyi was not reported before the current study.

Compound 7 was isolated as white crystallineneedles. EIMS for C32H52O2 m/z (rel. int.): 468 [M+](17.2%), 453 (2.9%), 425 (1.5%), 408 (1.7%), 365(3.9%), 189 (75.4%), 109 (73.1%), 43 (100%). Thediagnostic peaks occurred at m/z 408 [M-AcOH],249 [M-C16H27] and 189 [249-AcOH]. The IR spec-trum of compound 7 exhibited characteristic absorp-tion frequency at 1732 cm-1 (C=O), 3073 cm-1

(exomethylene group), 2939 cm-1 (C-H) and 1244cm-1 (C-O). The 1H-NMR spectrum of compound 7showed eight methyl resonances, seven of thembetween δH 0.76-1.66 ppm. The 1H-NMR spectrumof compound 7 was similar to compound 6 withaddition of an acetate methyl resonance at δH 2.01ppm (H-32) and the proton resonance at δH 4.46 ppm(H-3) which is further downfield due to presence ofthe acetoxy group. Based on spectral informationand in comparison with that in the literature (17)compound 7 was identified as lupeol acetate. Lupeol

acetate has never been reported isolated before fromF. burtt-davyi.

Compound 8 was isolated as a light brownpowder. The mass spectrum of compound 8

obtained by HR-ESI-MS (positive mode) gave m/zvalue at 313.0685 corresponding to the molecularformula (C15H14O6) [M + Na]+, (calc. C15H14O6 Na313.2596). The IR spectrum showed characteristicabsorption bands for the O-H group (3215 cm-1),C=C group (1623 cm-1), and C-O group (1145ñ1019cm-1). The 1H-NMR spectrum of compound 8

showed characteristic resonances for flavonoids atδH 6.85 (H-2í), 6.76 (H-5í) and 6.74 (H-6í) ppmfrom the B-ring catechol moiety as well as at δH 5.94(H-6) and 5.87 (H-8) ppm from the meta-coupledprotons of the A-ring resorcinol moiety. The protonresonances at δH 4.59 (H-2), 3.99 (H-3), 2.87 (H-4)and 2.53 (H-4) ppm were used to establish a flavanolskeleton for compound 8. The 13C-NMR spectrumshowed a diagnostic peak at δC 82.8 (C-2) ppm aswell as a strong H-2/H-3 correlation from the COSYexperiment, thus confirming the structure of com-pound 8 to be (+)-catechin (18). Catechin and itsanalogues have been shown to possess significantbioactivity, such as anti-inflammatory, anti-allergic,anti-mutation, antioxidant and anti-aging activity(19). This is the first report of compounds 1-8 beingreported in F. burtt-davyi.

Total phenolic content (TPC)

The extraction yields indicated that the stembark of F. burtt-davyi produced the highest amountof extract. Furthermore, the total phenolic content(TPC) of the MeOH extract of the stem bark wassignificantly higher (p < 0.05) than that from otherextracts (Table 1). Based on this result, the MeOHextracts from the leaves and stem barks were select-ed for in vitro antioxidant study.

Table 1. Total phenolic content (TPC) and extraction yield (%, mg extract per g sample ◊ 100) of F. burtt-davyileaves and stem bark extracts.

Extracts Yield (%) TPC (mg/g GAE) *

Leaves

EtOAc 0.51c 0.43 ± 0.01c

MeOH 3.05b 1.14 ± 0.12b

Stem bark

EtOAc 0.80c 1.05 ± 0.22bc

MeOH 5.46a 5.90 ± 0.45a

Data are presented as the mean ± SD (n = 3). *GAE (gallic acid equivalent).Values with different superscriptsletters along a column are significantly different from each other by Tukeyís HSD multiple post hoc test, (p <0.05).


DPPH free radical-scavenging activity assay

Most antioxidants possess proton radical scav-enging activity which may be monitored by discol-oration of the purple 1,1-diphenyl-2-picrylhydrazyl(DPPH) radicals to yellow DPPH in a dose-depend-ent pattern. For this study, the DPPH assay was usedto detect the antioxidant activity of the extracts andcompounds from F. burtt-davyi as shown in Figure 1.The results indicated that at lower concentrations (≤25 µg/mL), the activity of the extracts (stem bark andleaves) and compounds were comparable but weresignificantly lower than that of ascorbic acid (AA).

At higher concentrations (≤ 125 µg/mL), theactivity of the MeOH extract from the bark, (+)-cat-echin and AA were not significantly different buthigher than the other substances tested. The highactivity of the MeOH extract of the bark could beattributed to the presence of (+)-catechin in theextract, thereby suggesting that the activity of anextract may be as a result of the presence of onebioactive compound, which, at lower concentra-

tions, may be diminished by the antagonistic effectsof other compounds or the presence of other non-active compounds.

Ferric reducing antioxidant power assay (FRAP

assay)

Numerous studies have linked the electrondonation capacity (reduction) of an antioxidant to itsantioxidative activity (20). The presence of electrondonating substances, such as antioxidant samples,causes the reduction of the Fe3+/ferricyanide com-plex to the ferrous form (Fe2+). The total reducingpower of the extracts and compounds from F. burtt-davyi were compared to AA (Fig. 2).

Similar to the results produced by the DPPHradical scavenging activity, the results obtainedfrom the FRAP assay showed the reducing power ofthe MeOH extract from the stem bark, (+)-catechinand AA to be comparable and significantly higherthan that of the MeOH extract from leaves and pheo-phytin a.

Figure 2. DPPH radical scavenging activity of MeOH extracts of F. burtt-davyi stem bark (B), leaves (L), pheophytin a, (+)-catechin andascorbic acid (AA). Different letters for a concentration indicate significantly different means (Tukeyís post hoc comparisons, p < 0.05)

Table 2. IC50 of different extracts and compounds from F. burtt-davyi for various antioxidant assays.

Extracts/compound DPPH* (µg mL) FRAP* ( µg/mL) H2O2 scavenging* ( µg/mL)

L(MeOH) 405.39 ± 20.58c 420.95 ± 8.46c 449.56 ± 6.66c

B(MeOH) 58.28 ± 5.05b 46.09 ± 0.06b 151.03 ± 1.6b

Pheophytin a 611.15 ± 10.06d 777.80 ± 1.60d > 1000d

(+)-Catechin 61.19 ± 0.68b 46.89 ± 0.33b 125.15 ± 1.16b

Ascorbic acid (AA) 1.14 ± 0.08a 39.32 ± 0.017a 81.56 ± 0.63a

Each value is represented as the mean ± SD (n = 3). a-d Means in the same column followed by a different letter are significantly different(p < 0.05). *No significant difference between assays (p > 0.05).


Hydrogen peroxide-scavenging activity assay

Although hydrogen peroxide is a weak oxidiz-ing agent, it can cross cell membranes rapidly andwhile inside the cell, form hydroxyl radicals whichare primarily responsible for its toxic effects.Therefore, removal of the hydroxyl free radical isnecessary to ensure a good health status. The scav-enging effects of the extracts and isolated com-pounds in F. burtt-davyi were evaluated against thisfree radical (Fig. 3). The antioxidant activity of theextracts and compounds using hydrogen peroxidewere similar to those produced by the DPPH andFRAP assays thereby confirming the activities of allthe tested substances.

Table 2 shows the IC50 values for the differentantioxidant assays. Although compounds 1-8 weretested for antioxidant activity, the IC50 values forsitosterol, campesterol, stigmasterol, lutein, lupeol

and lupeol acetate were extremely high (> 1000µg/mL) for all assays, therefore, their results are notshown in Table 2. The results confirm that theMeOH extract and (+)-catechin have a significantlyhigher antioxidant activity (p < 0.05), with compa-rable IC50 values for all three antioxidant assays.However, the IC50 value for the standard AA wassignificantly lower than the plant extracts and com-pounds. From the TPC and antioxidant activityresults, it is clear that higher TPC indicates higherantioxidant activity. This is in agreement with pre-vious reports (21, 22).

Thus, the present study has shown that theleaves and stem bark of F. burtt-davyi possess mod-erate to significantly good antioxidant activity andmay contribute to the retardation of the inflammato-ry process mediated by reactive oxygen metabolitesfrom phagocytic leukocytes that invade the tissues

Figure 3. Reducing power of MeOH extracts of F. burtt-davyi stem bark B (MeOH), leaves L (MeOH), isolated compounds (pheophytina and (+)-catechin) and ascorbic acid (AA). Values represented as the mean ± SD (n = 3)

Figure 4. Hydrogen peroxide scavenging activity of MeOH extracts of F. burtt-davyi stem bark B (MeOH), leaves L (MeOH), isolatedcompounds (pheophytin a and (+)-catechin) and ascorbic acid (AA). Values represented as the mean ± SD (n = 3)

3

2.5

2

1.5

1

0.5


and cause injury to essential cellular components(23). In addition, previous studies have shown thatplant-derived sterols exhibit anti-carcinogeniceffects on different types of cancers (24-27). F burtt-davyi could therefore be said to possess anti-cancerpotential having isolated several sterols from thestem bark.

CONCLUSION

To the best of our knowledge, this is the firstreport of the phytochemical constituents of F. burtt-davyi, resulting in the isolation of a carotenoidwhich has been documented to exhibit moderateantibacterial activity and pheophytin a with stronganti-HCV-NS3 protease activity with little cytotoxi-city. The data from this study suggest that F. burtt-davyi possessed moderate to good antioxidativeactivity and can be used as a potential alternativemedicine for oxidative stress related non-communi-cable chronic diseases.

Acknowledgments

The authors are grateful to the College ofAgriculture, Engineering and Science, UKZN forsupport.

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Received: 24. 11. 2015


Traditional system of medicines or herbal careis the original source for most of the medicines andis used worldwide. Medicinal plants possess a widevariety of chemical constituents which are helpfulfor curing different diseases. Recent discovery ofmedicinal plants have led to improvement of humanhealth care systems. Plant based drugs are used asmedicines either as a whole or their principle con-stituents are separated by different chemical tech-niques. This practice continues because of differentbiological advantages, pharmacological activitiesand fewer side effects associated with medicinalplants (1).

Inflammation is a primary defense mechanismthat helps the body to protect itself against varioustypes of diseases, allergens, chemicals and othertoxic reactions. Inflammation is a process whichinvolves a large variety of inflammatory mediatorsand prominent increase in localized leucocytes.Prostaglandins are the substances that modulate thecell and tissue response during inflammation. Their

biosynthesis also continues during the cardiovascu-lar disease, cancers and colonic adenomas. Althoughit is a mechanism of protection for the body, anuncontrolled inflammation and various mediators ofinflammation can induce or maintain a disease andcan even aggravate it (2). Plants are widely used fortreating inflammation because the adverse effectsproduced by these plant based drugs are very few. Inaddition, they are easily affordable and lower in costthan the synthetic drugs (3).

Pyrexia or fever occurs as bodyís naturaldefense mechanism in response to any infection ordisease. In pyrexia, an environment is created insidethe body in which the infectious pathogens and dam-aged tissues are unable to survive (4). Normal bodytemperature is maintained and regulated by hypo-thalamus that maintains a balance between heat lossand production. Fever occurs due to some distur-bances in this thermostat of body and as a resultbodyís temperature rises. The temperature regulat-ing mechanisms then works to normalize the body

EVALUATION OF ANTI-INFLAMMATORY, ANALGESIC AND ANTIPYRETIC ACTIVITIES OF AQUEOUS AND ETHANOLIC EXTRACTS OF

SEEDS OF BUCHANANIA LANZAN SPRENG. IN ANIMAL MODELS

AMIR MEHMOOD1, IRFAN HAMID2, ALI SHARIF1, MUHAMMAD FURQAN AKHTAR1, BUSHRA AKHTAR3, AMMARA SALEEM4, JAVED IQBAL1, MARYAM SHABBIR1

and SAJID ALI1*

1Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan2Akson College of Health Sciences, Mirpur University of Science and Technology, AJK, Pakistan

3Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan4Department of Pharmacy, Government College University, Faisalabad, Pakistan

Abstract: The present study was designed to evaluate the anti-inflammatory, analgesic and antipyretic activi-ties of the aqueous and ethanolic extracts of seeds of Buchanania lanzan Spreng. Albino mice were used asexperimental animals to evaluate these activities. The study was performed in three phases; Phase-I for evalu-ation of anti-inflammatory activity, Phase-II for antipyretic and Phase-III for analgesic activities were evaluat-ed. Carrageenan induced paw edema, brewer yeast induced pyrexia and acetic acid induced writhing methodswere used to evaluate anti-inflammatory, antipyretic and analgesic activities, respectively. Tests were per-formed by dividing the animals in five groups. First group was negative control, second group was positive con-trol, third, fourth and fifth groups were treated with 125, 250 and 500 mg/kg of extracts. respectively. The datawere statistically analyzed using ANOVA where p < 0.05 were considered significant. The results suggestedthat seeds of Buchanania lanzan Spreng. possess anti-inflammatory, analgesic and antipyretic activity.

Keywords: Buchanania lanzan Spreng., analgesic, anti-inflammatory, antipyretic

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* Corresponding author: e-mail: [email protected]; phone: 0092-333-4404880

1602 AMIR MEHMOOD et al.

temperature. These mechanisms include dilation ofsuperficial blood vessels and sweating etc. Whenany infectious agent enters into the body through abreak in its barriers, it will interact with immunecells and will promote the release of endogenousmediators like cytokines, prostaglandins andendothelins. In pre-optic area of anterior hypothala-mus, PGE2 play a vital role in fever induction (5).

Pain is a sensorial modality and sometimes it isthe only feature for diagnosis of various diseases.Many forms of therapy are available for curing thiscondition, among them medicinal herbs are widelyused (6). Pain is a sensation which is unpleasant andis localized to a part of body. In response to tissueinjury visceral distensions, peripheral nociceptorsand some other factors are stimulated, which devel-

op pain. Perception of pain is a normal physiologi-cal response and it is mediated by the healthy nerv-ous system (7).

Different experimental models are used for eval-uating anti-inflammatory, analgesic and antipyreticactivities of herbal plants. For studying anti-inflam-matory activity, a large number of experimental mod-els have been developed. These models are basicallyof two types; acute inflammatory model and chronicinflammatory model. Acute models are developed tostudy and evaluate the drugs which are involved inerythema, leucocyte migration and chemotaxis,changes in vascular permeability, phagocytosis-poly-morphonuclear leucocytes, measurement of localpain and rat paw edema (8). Acute-inflammatorymodels are carrageenan induced paw edema, xylene

Table 1. Experimental design.

Groups Drug Dose Route No. of animals

Group 1 (negative control) Normal saline 1 mL/kg Oral 5

Group 2 (positive control) Indomethacin 10 mg/kg Oral 5

Group 3 Plant extract 125 mg/kg Oral 5



Table 2. Anti-inflammatory activity of Buchanania lanzan Spreng. (aqueous extract).

Average paw volume (mm)Groups

1 h 2 h 3 h

Normal saline 3.70 ± 0.01 3.76 ± 0.01 3.97 ± 0.01

Indomethacin 2.93 ± 0.03 2.13 ± 0.01 1.91 ± 0.01

125 mg/kg 3.41 ± 0.02 3.16 ± 0.05 2.86 ± 0.05

250 mg/kg 3.37 ± 0.02 2.94 ± 0.01 2.48 ± 0.04

500 mg/kg 3.25 ± 0.41 2.71 ± 0.01 2.39 ± 0.08

Note: All specified doses showed a statistically significant (p < 0.05) effect.

Table 3. Percentage inhibition of carrageenan induced paw edema (aqueous extract).

Inhibition of carrageenan Average Groups induced paw edema (%) inhibition

1 h 2 h 3 h (%)

Indomethacin 20.8 43 51.8 38.2

125 mg/kg 7.8 15.9 27.9 17.2

250 mg/kg 8.9 21.8 37.5 22.7

500 mg/kg 12.1 27.9 39.7 26.5

Evaluation of anti-inflammatory, analgesic and antipyretic activities of... 1603

induced ear edema, egg albumin induced edema,arachidonic acid induced ear edema and croton oilinduced edema in mice/rats. Chronic models are cot-ton pellet induced granuloma and adjuvant arthritis inmice/rats (9). Analgesic activity is determined in twoways; central analgesic activity and peripheral anal-gesic activity. Peripheral analgesic activity is evaluat-ed by acetic acid induced writhing and hot platemethod (10). Antipyretic activity is studied by brew-erís yeast induced pyrexia, vaccines induced pyrexiaand D-amphetamine induced pyrexia (11).

Buchnania lanzan Spreng. (family Anacardia-ceae) is commonly known as Char, chironji,Almondette Tree, Murkali, Nurkale. It is an indige-nous Hill Plant, Plain Land commonly foundthroughout the greater part of India in dry deciduousforests up to an altitude of 1200 m. The decoction ofits bark is useful in abdomen disease, cough andbronchitis. Fruit is used to treat nervous debility,cardiac debility, constipation, fever, ulcer,oligospermia and general debility (12). The parts ofplant like roots, leaves, fruits, seeds and gum areused for various medicinal applications like cure forblood disorder, fever, ulcer, burning sensation ofbody, diarrhea, dysentery, asthma and snakebite(13). Aqueous and ethanolic extracts of seeds ofChar were used to evaluate anti-inflammatory, anal-gesic and anti-pyretic activity.

The present study was undertaken to assess theanti-inflammatory, antipyretic and analgesic activity

of aqueous and ethanolic extracts of seeds ofBuchnania lanzan Spreng.

MATERIAL AND METHOD

Collection of plant material

Seeds of Buchnania lanzan Spreng. were pur-chased from local market of Lahore, Pakistan. It wasidentified and authenticated by Prof. Dr.Zaheerudin, Botany Department of GovernmentCollege University, Lahore, Pakistan. The seedswere preserved in herbarium of GovernmentCollege University, Lahore, Pakistan.

Washing, drying, grinding and storage of plant

The seeds were washed with distilled water toremove dirt particles and contamination. Washedseeds were dried at room temperature under shadefor appropriate time period. Fully dried seeds werethen grinded with mortar and pestle until a coarsepowder was obtained. The powder was passedthrough sieve #20 and stored in air tight, labeledcontainers till further processing.

Extract preparation

Aqueous and ethanolic extracts of Buchnanialanzan Spreng. seedís powder were prepared bytriple maceration process. For the preparation ofaqueous extract, 500 g of powdered material wassoaked in 1000 mL of distilled water in an amber

Table 5. Percentage inhibition of carrageenan induced paw edema (ethanolic extract).

Inhibition of carrageenan Average Groups induced paw edema (%) inhibition

1 h 2 h 3 h (%)

Indomethacin 7.4 47.0 51.2 41.8

125 mg/kg 13.0 26.0 30.0 23.0

250 mg/kg 16.0 35.0 40.0 30.3

500 mg/kg 21.0 36.0 46.0 34.3

Table 4. Anti-inflammatory activity of Buchanania lanzan Spreng. (ethanolic extract).

GroupsMean values of paw edema (mm) ± SEM

1 h 2 h 3 h

Normal saline 3.72 ± 0.01 3.88 ± 0.01 3.98 ± 0.01

Indomethacin 2.70 ± 0.07 2.10 ± 0.01 1.94 ± 0.01

125 mg/kg 3.21± 0.01 2.84 ± 0.01 2.78 ± 0.01

250 mg/kg 3.11 ± 0.01 2.52 ± 0.01 2.38 ± 0.01

500 mg/kg 2.93 ± 0.01 2.48 ± 0.01 2.14 ± 0.04



colored glass container for 3 days. After stated time,the solvent was removed using muslin cloth and theremaining plant material was again soaked in 500mL of distilled water and whole process was repeat-ed thrice. Ethanolic extract was prepared by thesame method using ethanol as solvent.

Drying of extracts

The extracts were filtered through Whatmanfilter paper and then dried in rotary vacuum evapo-rator until a semi-solid mass was obtained. Thissemi-solid mass was spread in Perti dishes to com-pletely dry the extract in oven. The dried extractswere stored in refrigerator at 4OC until further pro-cessing.

Grouping of animals

Albino male mice were obtained fromDepartment of Theriogenology, University ofVeterinary and Animal Sciences, Lahore, Pakistan.The weight of each mouse ranged in 20-25 g. Theanimals were of the same breed and batch. The micewere divided into 5 groups with each group havingfive mice. They were kept in polycarbonate cages

under controlled temperature (22-25OC) and had freeaccess to feed and water.

Pharmacological evaluation

Pharmacological evaluation of the seedextracts of Buchanania lanzan Spreng. was done inthree phases. In phase-I anti-inflammatory activitywas evaluated. In phase-II analgesic activity and inphase-III anti-pyretic activity were evaluated. Thetreatment strategy for animal groups is given inTable 1.

Phase-I - Evaluation of anti-inflammatory activityInflammation was induced by injecting 1% car-

rageenan solution (dose of 0.05 mL) to the right hindpaw of mice. Test drug and standard drug were given1 h before the administration of carrageenan inrespective groups. In negative control group, themice were treated with normal saline and in positivecontrol, indomethacin was given. A mark was madeat the paw of each mouse up to the ankle joint. Pawvolume was measured up to ankle joint in drug treat-ed and drug untreated groups before and after admin-istration at a time interval of 0, 1, 2 and 3 h (14).

Table 6. Brewer's yeast induced antipyretic activity (aqueous extract).

Mean values of temperature (∞F) ± SEM

Groups Before drug administration After drug administration

Normal temp. 18 h 1 h 2 h 3 h

Normal saline 97.51 ± 0.01 102.95 ± 0.03 102.14 ± 0.02 102.78 ± 0.04 102.98 ± 0.01

Indomethacin 97.58 ± 0.09 102.71 ± 0.03 101.34 ± 0.04 100.31 ± 0.09 97.02 ± 0.01

125 mg/kg 97.96 ± 0.02 102.38 ± 0.08 101.86 ± 0.01 99.73 ± 0.01 99.04 ± 0.01

250 mg/kg 96.85 ± 0.03 102.74 ± 0.02 101.43 ± 0.01 99.34 ± 0.01 98.55 ± 0.02

500 mg/kg 97.20 ± 0.10 102.12 ± 0.02 101.14 ± 0.03 99.03 ± 0.03 98.19 ± 0.19


Table 7. Brewer's yeast induced antipyretic activity (ethanolic extract).

Mean values of temperature (∞F) ± SEM

Groups Before drug administration After drug administration

Normal temp. 18 h 1 h 2 h 3 h

Normal saline 97.52 ± 0.07 102.83 ± 0.27 102.04 ± 0.01 102.84 ± 0.01 102.96 ± 0.01

Indomethacin 97.72 ± 0.04 102.73 ± 0.03 101.50 ± 0.05 100.37 ± 0.03 97.23 ± 0.04

125 mg/kg 97.26 ± 0.02 102.54 ± 0.01 101.96 ± 0.01 100.73 ± 0.01 99.74 ± 0.01

250 mg/kg 97.15 ± 0.03 102.32 ± 0.02 101.44 ± 0.01 100.34 ± 0.01 99.56 ± 0.02

500 mg/kg 96.94 ± 0.03 102.34 ± 0.02 101.64 ± 0.03 100.03 ± 0.03 99.12 ± 0.12



Quantification of anti-inflammatory activityThe anti-inflammatory activity of the seed

extract of Buchanania lanzan Spreng. was quantifiedby measuring the paw edema (in mm) using digitalVernier caliper. The results were expressed in termsof mean values of paw edema of each group ± SEM.

Reduction in paw edema was measured by thepercentage inhibition of the positive control andextract treated groups as compared to the negativecontrol. The percentage inhibition was calculatedusing the following formula:

Percentage inhibition = (Control ñ Treated) /(Control) ◊ 100

Phase-II - Evaluation of antipyretic activityBrewerís yeast solution was used to induce

fever in mice by subcutaneous administration ofsolution below the nape of the neck. Initial tempera-ture was recorded by using digital clinical ther-mometer. After 18 h of administration, animal whichshowed a mean rise of 0.3-0.5OC body temperaturewere selected. Animals were treated with normalsaline, standard drug and drug extracts as per exper-iment design. Temperature of mice was recordedafter an interval of 1, 2 and 3 h post dosing (15).

Quantification of antipyretic activityAntipyretic activity was quantified by check-

ing the decrease in body temperature at different

time intervals. The results were calculated in F ±SEM for all groups.

Phase-III - Evaluation of analgesic activityAcetic acid induced writhing method was used

to evaluate analgesic activity of Buchanania lanzanSpreng. seed extract. The mice were treated withintraperitoneal injection of 1% acetic acid solution(0.1 mL) and number of writhing movements wascounted for 20 min (16).

Quantification of analgesic activityThe reduction in number of writhing after

administering positive control and drug extractswith respect to negative control group was quanti-fied by calculating percentage inhibition using fol-lowing formula:

Percentage inhibition = (Nc ñ Nt) / (Nc) ◊ 100where, Nc is mean number of writhing in controlgroup, Nt is mean number of writhing in treatedgroup


For statistical analysis, the data were evaluatedusing Statistical Package of Social Sciences (SPSS).The results were expressed as the mean ± SEM andanalysis of variance (ANOVA) was applied to thedata.

Table 8. Acetic acid induced analgesic activity of Buchanania lanzan Spreng. (aqueous extract).

Groups Drug DoseNumber of writhing Inhibition of

(mean) ± SEM writhing (%)

Group 1 Normal saline 1 mL/kg 66.8 ± 0.44 N/A

Group 2 Indomethacin 10 mg/kg 17.6 ± 0.54 73.65

Group 3 Test extract 125 mg/kg 44.4 ± 0.54 33.53




Table 9. Acetic acid induced analgesic activity of Buchanania lanzan Spreng. (ethanolic extract).

Groups Drug DoseNumber of writhing Inhibition of

(mean) ± SEM writhing (%)

Group 1 Normal saline 1 mL/kg 65.0 ± 0.70 N/A

Group 2 Indomethacin 10 mg/kg 16.8 ± 0.83 74.15







In previous literature, phytochemical investi-gation revealed the presence of new glycoside,myricetin-3-rhamnoside-3-galactoside (17). Thecomponent fatty acids of the seed fat were found tobe myristic, palmitic, stearic, oleic and linoleicacids. The moisture, crude protein, pentosan andwater soluble mucilage contents of seeds werereported as 5.2, 6.95, 3.8 and 2.8 percent, respec-tively. The mucilage showed absence of protein andpentosan (18).

The results of anti-inflammatory effect ofaqueous and ethanolic extract of Buchanania lanzanSpreng. on carrageenan-induced edema in paw ofmice are presented in Tables 2 and 4, respectively,whereas percentage inhibition by aqueous and

ethanolic extract is depicted in Tables 3 and 5,respectively. Edema development due to inflamma-tion is a biphasic event in which the initial phase isattributed to the release of histamine and serotonin.The second phase of edema is due to the release ofprotease, prostaglandins and lysosomes. Most of theanti-inflammatory drugs are clinically effective insecond phase of inflammation (19). The data repre-sented that the ethanolic extract at dose of 500mg/kg showed significant reduction (p < 0.01) inedema and faster rate of inhibition as compared toother doses. However, the aqueous and ethanolicextracts showed moderate reduction in edema whencompared with the extract of standard drug (Fig. 1).The carrageenan induced paw edema model isknown to be sensitive to the effect of NSAIDs whichprimarily inhibits cyclooxygenase involved in syn-

Figure 1. Percentage inhibition of carrageenan induced paw edema by standard drug, aqueous and ethanolic extract of Buchanania lanzanSpreng.

Figure 2. Percentage inhibition of Brewerís yeast induced pyrexia by standard drug, aqueous and ethanolic extract of Buchanania lanzanSpreng.


thesis of prostaglandins (20). Therefore, it can bereasonably concluded that the inhibitory effect ofaqueous and ethanolic extract of Buchanania lanzanSpreng. on inflammation in mice may be due to theinhibition of cyclooxygenase enzyme.

The antipyretic activity of aqueous and ethano-lic extract is given in Tables 6 and 7, respectively. Anincrease in temperature was evident after 18 h ofBrewerís yeast administration. After the administra-tion of indomethacin and drug extracts, a significantdecrease in temperature was noted. The extractsshowed dose dependant decrease in temperature withincreasing potency from 125 to 500 mg/kg. The aque-ous extract of seed at dose of 500 mg/kg had evidentantipyretic activity but it was moderate as comparedto the positive control after 3 h of drug administration.Therefore, aqueous extract proved to contain moreantipyretic activity as compared to ethanolic extractbut lesser than standard antipyretic drug (Fig. 2).

The peripheral analgesic activity of drugextract was tested by acetic acid induced writhingtest. Acetic acid induced writhing test is a standardtest to check the pain sensitivity of opiates and non-opiates analgesics. The pain sensation is due toabdominal constriction due to irritation of peritonealcavity caused by acetic acid. Prolonged irritationleads to an increase in level of prostaglandins (PGE2

and PGF2α) biosynthesis and lipoxygenase productsin peritoneal fluids. The increase in these productsenhances the level of free arachidonic acid whichsubsequently develops inflammatory pain byincreasing capillary permeability in peritoneal cavi-ty of abdomen (21). The reference drug used wasindomethacin which is non-opiate analgesic. ThisNSAID involves blockade of production of

prostaglandins by inhibition of both COX-1 andCOX-2. The aqueous and ethanolic seed extractproduced a significant decrease in the writhingcounts (Tables 8 and 9). Minimum number ofwrithing was observed with 500 mg/kg ethanolicextract. The percentage inhibition of pain inducedby acetic acid was dose dependent i.e., with anincrease in dose of aqueous and ethanolic extract thenumber of writhing in groups decreased in contrastto negative control group (Fig. 3). Analgesic activi-ty of aqueous and ethanolic seed extract indicatesthat peripherally active analgesic property might bepresent (p < 0.01).

CONCLUSION

The result of biological screening on the frac-tions from aqueous and ethanolic extract ofBuchanania lanzan Spreng. confirms consistentactivities in anti-inflammatory, antipyretic and anal-gesic tests.

REFERENCES

1. Sharma J., Srivastava A., Thakur S., Barpete P.,Singh S.: Int. J. Pharm. Life Sci. (IJPLS) 1, 18(2010).

2. Vinay J., Panigrahi M.: J. Pharm. Res. 5,(2012).

3. Ravi V., Saleem T. M., Patel S., RaamamurthyJ., Gauthaman K.: Int. J. Appl. Res. Nat. Prod.2, 33 (2009).

4. Chattopadhyay D., Arunachalam G., Ghosh L.,Rajendran K., Mandal A., Bhattacharya S.: J.Pharm. Pharm. Sci. 8, 558 (2005).

Figure 3. Percentage inhibition of acetic acid induced pain by standard drug, aqueous and ethanolic extract of Buchanania lanzan Spreng.


5. Dos Santos M.D., Almeida M.C., Lopes N.P.,De Souza G.E.P.: Biol. Pharm. Bull. 29, 2236(2006).

6. Almeida R., Navarro D., Barbosa-Filho J.:Phytomedicine 8, 310 (2001).

7. Freeman D. L.: JAMA 286, 971 (2001).8. Barbosa-Filho J.M., Piuvezam M.R., Moura

M.D., Silva M.S., Lima K.V.B. et al.: RevistaBrasileira de Farmacognosia 16, 109 (2006).

9. Gupta M., Mazumdar U.K., Sivakumar T.,Vamsi M.L. M., Karki S.S et al.: Biol. Pharm.Bull. 26, 1342 (2003).

10. Bhaskar V., Balakrishnan N.: DARU J. Pharm.Sci. 17, 168 (2009).

11. Okokon J.E., Nwafor P.A.: Pak. J. Pharm. Sci.23, 385 (2010).

12. Mishra R.K., Patel S.P., Srivastava A.,Vashistha R.K., Singh A., Puskar A.K.: Nat.Sci. 10, 22 (2012).

13. Malik S., Chaudhury R., Panwar N., DhariwalO., Choudhary R., Kumar S.: Genet. Resour.Crop Ev. 59, 615 (2012).

14. Adedapo A.A., Sofidiya M.O., Maphosa V.,Moyo B., Masika P.J., Afolayan A.J.: Rec. Nat.Prod. 2, 46 (2008).

15. Pradhan D.K., Jana G.K., Panda S., ChoudhuryN.K., Patro V.J.: Drug Invention Today. 1, 29(2009).

16. Purnima A., Koti B., Tikare V., Viswanathas-wamy A., Thippeswamy A., Dabadi P.: IndianJ. Pharm. Sci. 71, 461 (2009).

17. Nasim K., Arya R., Babu V., Ilyas M.:Phytochemistry 31, 2569 (1992).

18. Sengupta A., Roychoudhury S.K.: J. Sci. FoodAgr. 28, 463 (1977).

19. Olajide O.A., Awe S.O., Makinde J.M., EkhelarA.I., Olusola A. et al.: J. Ethnopharmacol. 71,179 (2000).

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Received: 5. 12. 2015


Fibrinogen (Fg) - the precursor of a fibrin clot,is an abundant, multifunctional protein constitutingapproximately 4% of the total plasma protein (1-3).Fibrinogen not only plays an important role in bloodcoagulation, but also acts as an adhesive proteinessential for blood platelet adhesion and aggregation(4, 5).

Recently, it has been shown that homocysteine(Hcy) and its metabolites, including homocysteinethiolactone (HTL), mediate the modifications (N-homocysteinylated or S-homocysteinylated pro-teins) of various hemostatic proteins, including:plasma proteins (i.e., fibrinogen) and blood plateletproteins (6-8). The modifications of these hemostat-ic proteins induced by Hcy or its derivatives seem tobe the main reason for biotoxicity of homocysteinein cardiovascular system and cardiovascular dis-eases (6-8). Our earlier studies reported that modifi-cations of fibrinogen induced by Hcy or its thiolac-

tone may be associated with changes in hemostasis,including the coagulation process (8) and bloodplatelet activation - platelet adhesion (9, 10).Increased concentration of total Hcy (> 15 µM) inplasma is called hyperhomocysteinemia. The aim ofour study was to establish the influence of commer-cial phenolic extract - extract from berries of Aroniamelanocarpa (AronoxÆ), on the changes of adhe-sive properties of fibrinogen in in vitro model ofhyperhomocysteinemia (induced by Hcy and HTL).A. melanocarpa grows in many sites throughoutEurope, has no special soil requirements or fertiliza-tion needs, is very resistant to pests (no chemicalsprayers are needed to have healthy fruits), has fruitevery year regardless of conditions, and produceslarge amounts of polyphenol (including antho-cyanins) rich fruits. There are no data in the litera-ture indicating toxic action of A. melanocarpa fruits,juice, extracts, or other aronia products, including

COMMERCIAL EXTRACT FROM ARONIA AS A MODULATOR OF ADHESIVE PROPERTIES OF FIBRINOGEN TREATED WITH

HOMOCYSTEINE AND ITS THIOLACTONE IN VITRO

BEATA OLAS1*, BOGDAN KONTEK1, WIES£AW OLESZEK2 and ANNA STOCHMAL2

1Department of General Biochemistry, Faculty of Biology and Enviromental Protection, University of £Ûdü, Pomorska 141/143, 90-236 £Ûdü, Poland

2Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100 Pu≥awy, Poland

Abstract: Research has confirmed the positive effect of berries of Aronia melanocarpa on the cardiovascularsystem. The protective effects of polyphenol-rich extract from berries of A. melanocarpa against changes in bio-logical properties of fibrinogen were studied. In in vitro model of hyperhomocysteinemia the capability of fib-rinogen to interact with human blood platelets was measured by platelet adhesion in the presence of extractfrom A. melanocarpa. We induced hyperhomocystenemia using a reduced form of homocysteine (Hcy, at a finalconcentration of 0.01, 0.1 and 1 mM) and the most reactive form of Hcy ñ its cyclic thioester, homocysteinethiolactone (HTL, at a final concentration of 0.1, 0.5 and 1 µM). It was observed that Hcy or HTL-treated fib-rinogen, in comparison with untreated molecule, had a distinct capability to mediate blood platelet adhesion.The experiments also indicate that polyphenol-rich extract from black chokeberries (at final concentrations of2.5ñ10 µM/mL) reduced the toxic action of Hcy and HTL on the adhesive properties of fibrinogen. The possi-ble protection exerted by black chokeberry extract, through restoring the platelet adhesion of Hcy or HTL treat-ed fibrinogen, may be important for vascular diseases.

Keywords: aronia, fibrinogen, blood platelets, homocysteine, hyperhomocysteinemia

Abbreviations: fg ñ fibrinogen, Hcy ñ homocysteine, HTL ñ homocysteine thiolactone

1609

* Corresponding author:e-mail: [email protected]

1610 BEATA OLAS et al.

AronoxÆ. Moreover, research has confirmed thepositive effect of berries of A. melanocarpa on thecardiovascular system (11). AronoxÆ is also richsource of valuable phytochemicals that are responsi-ble for the biological action of this extract (i.e., anti-platelet or antioxidative activity).


Chemicals

Reduced forms of D,L-homocysteine and D,L-homocysteine thiolactone were purchased fromSigma (St Louis, MO). Thrombin was purchasedfrom Biomed (Lublin, Poland). The natural concen-tration of total Hcy in human plasma was 8ñ14 µM(12).

Biological materials

Aronia melanocarpa is grown in Poland atlarge plantations to be used to produce phenolic richjuice, jams and phenolic rich extracts. The materialused for phenolic rich extract production came fromcommercial production of aronia berries. HPLC sep-aration of phenolic rich extracts from berries of aro-nia was described previously (13, 14). The total con-centration of phenolics in the phenolic-rich powderused in this study was 309.6 mg/g of extract (includ-ing phenolic acids (isomers of chlorogenic acid) ñ149.2 mg/g of extract, anthocyanins (anthocyaninglycosides: cyanidin 3-galactoside, cyanidin 3-glu-coside, cyanidin 3-arabinoside, cyanidin 3-xyloside)ñ 110.7 mg/g and flavonoids (quercetin glycosides)ñ 49.7 mg/g of extract) (14). Stock solutions of aro-nia extract (the commercial product ñ AronoxÆ byAgropharm Ltd., Poland; batch no. 020/2007k) weremade in H2O at a concentration of 5 mg/mL and keptfrozen until used for experiments.

Blood platelet and fibrinogen isolation

Blood samples were taken from 6 healthy vol-unteers not taking any medications or addictive sub-stances (including tobacco, alcohol and aspirin orany other anti-platelet drugs) and keeping a bal-anced diet (meat and vegetables), with similar socio-economic background, using no antioxidant supple-mentation. The protocol was passed by theCommittee for Research on Human Subjects of theUniversity of Lodz, number KBBN-U£/I/5/2011;and participants have signed written informed con-sent forms.

Fg was isolated from pooled citrated humanplasma by the cold ethanol precipitation techniquefollowed by ammonium sulfate fractionation at 26%saturation at 4OC, according to Doolittle et al. (15).

The concentration was determined spectrophoto-metrically at 280 nm using an extinction coefficient1.55 for a 1 mg/mL solution. The concentration ofpurified Fg in the reaction system was about 2mg/mL.

Human blood was collected into ACD solution(citric acid/ citrate/dextrose; 5 : 1, v/v) and bloodplatelets were isolated by differential centrifugationof blood as described by Wachowicz and KustroÒ(16). The platelets were counted by the photometricmethod according to Walkowiak et al. (17).

We induced hyperhomocysteinemia using areduced form of Hcy and the most reactive form ofHcy ñ its cyclic thioester, homocysteine thiolactone.Samples of human Fg (2 mg/mL) in 50 mMTris/HCl, 140 mM NaCl, pH 7.4 were exposed to:● D,L-homocysteine at a final concentration 0.01,

0.1 and 1 mM;● D,L-homocysteine thiolactone at a final concen-

tration 0.1, 0.5 and 1 µM;● the extract from berries of A. melanocarpa at a

final concentration between 2.5-10 µg/mL;● the extract from berries of A. melanocarpa at a

final concentration between 2.5-10 µg/mL and thereduced form of D,L-homocysteine at a final con-centration of 0.1 or 1 mM;

● the extract from berries of A. melanocarpa at afinal concentration between 2.5-10 µg/mL andD,L-homocysteine thiolactone at a final concen-tration of 1 µM.

Samples were incubated for 30 min at 37OC.The concentration of Hcy and HTL tested corre-sponds to levels found in human plasma duringhyperhomocysteinemia in vivo. Other authors usedvery similar model for inducing hyperhomocys-teinemia (i.e., blood samples were recovered for 10min prior to their supplementing with Hcy or HTL,each at the final concentration of 25 µM, correspon-ding to mild hyperhomocysteinemia) (18).

Blood platelet adhesion

Adhesion of blood platelets to Fg was deter-mined according to Tuszynski and Murphy (19).Wells of a 96-well microtiter dish (CLINIPLATEEB FB 50 PCS/CRS, Labsystems) were incubatedfor 2-3 h with 50 mL of Fg (dissolved in phosphate-buffered saline, pH 7.5 - PBS). The wells were aspi-rated, treated with 200 mL PBS containing 1% BSAfor 1 h, and then washed three more times with 200mL of PBS. Immediately after washing, the wellswere supplemented with 50 mL of the test agonist:thrombin (final concentration 0.1 U/mL) in PBS.Then, 100 mL of platelet suspension (4 ◊ 108

platelets/mL) was added to each well and the plate

Commercial extract from aronia as a modulator... 1611

was incubated at 37OC for 1 h. Non-adherent cellswere removed by aspiration and the wells werewashed three times with 200 mL of PBS. The totalcell-associated protein was determined by dissolv-ing the attached blood platelets directly in themicrotiter wells with 200 mL of the Sigma BCAworking solution, and incubated at 37OC for 60 min.Plates were allowed to cool to room temperature,cover sheets were removed, and the absorbance ofeach well determined at 540 nm with a microtiterplate reader (BioRad, Model 550). The absorbanceof control platelets (with native Fg) was expressedas 100%.

Data analysis

All the values in this study are expressed as themeans ± SD. The statistical analysis (to calculate theeffect of different concentrations of aronia extract)was performed using ANOVA and post hoc tests(Bonferroni). In order to eliminate uncertain data,the Q-Dixon test was performed.

RESULTS

In order to evaluate the altered functionality ofHcy- or HTL-modified Fg in the presence of aroniaextract, its ability to mediate platelet adhesion was

Figure 1A. The effect of aronia extract (2.5-10 µg/mL, 30 min, 37OC) on platelet adhesion of unstimulated platelets to microplate wellscoated with native fibrinogen and Hcy (0.1 mM, 30 min, 37OC)/HTL (1 µM, 30 min, 37OC) -treated fibrinogen

Figure 1B. The effect of aronia extract (2.5-10 µg/mL, 30 min, 37OC) on platelet adhesion of thrombin-stimulated platelets (B) to microplatewells coated with native fibrinogen (Fg) and Hcy (0.1 mM, 30 min, 37OC)/HTL (1 µM, 30 min, 37OC) -treated fibrinogen

1612 BEATA OLAS et al.

assessed. Our studies demonstrate that aronia extract(at all tested concentrations: 2.5, 5 and 10 µg/mL)did not change the ability of Fg to interact withblood platelets (by measuring platelet adhesion)(Fig. 1). On the other hand, as shown in Figure 1,both unstimulated (A) and thrombin-activatedplatelets (B) showed a reduced ability to adhere toHcy (0.1 mM) treated Fg. Unstimulated plateletsshowed a lower capacity to adhere to Hcy (0.1 mM)treated Fg than thrombin-activated platelets. Wealso demonstrate an inhibitory effect on plateletadhesion to modified Fg when Fg was treated withhomocysteine thiolactone (1 µM), and bloodplatelets were stimulated by thrombin (Fig. 1B). Theresults presented in Figure 1B demonstrate the pro-tective action of aronia extract on platelet adhesionto modified Fg when it was treated with plantextracts and Hcy or HTL, and blood platelets werestimulated by thrombin. Aronia extract had the sameproperties when we measured the binding of adher-ent resting platelets to Hcy-modified Fg (Fig. 1A).

DISCUSSION

Some results showed that homocysteine and itsthiolactone may induce the oxidative stress (20, 21).Other experiments indicate that elevated homocys-teine and folate deficiency have been associatedwith increased oxidative stress. Oxidant injury hasbeen proposed as a potential mechanism of athero-genesis in hyperhomocysteinemia (22). Results ofKolling et al. (23) demonstrated that supplementa-tion with folic acid can be used as an adjuvant ther-apy in cardiovascular alterations caused by Hcy.They observed that Hcy induces oxidative-nitrativestress in the heart of rats, but folic acid has protec-tive properties.

Epidemiologic studies suggest that the regularconsumption of polyphenolic antioxidants, second-ary metabolites of plants, is correlated with adecrease in the risk of cardiovascular disease, dia-betes, arthritis and cancer. Moreover, dietarypolyphenolic antioxidants can reduce oxidativestress stimulated by hyperhomocysteinemia (20, 22-30). Our earlier results (29, 31) also suggested thatchanges in the level of reactive oxygen species(including superoxide anion radicals in bloodplatelets), caused by plant phenolic extracts (aroniaextract and extract from grape seeds) may beresponsible for the inhibition of platelet activation(stimulated by thrombin) during hyperhomocys-teinemia. Kolodziejczyk et al. (32) observed thatgrape seed extracts reduced the biotoxicity action ofHcy and HTL on fibrinolysis.

Present experiments have demonstrated thatcommercial extract from berries of A. melanocarpa(AronoxÆ by Agropharm ñ used in our earlier tests(13, 14, 31) and by other authors (33-35)) reducesthe biotoxicity of Hcy and HTL on the adhesiveproperties of Fg (measured by the capability of Fg tointeraction with blood platelets), suggesting a possi-ble protective role in hyperhomocysteinemia. Therange of tested concentrations of aronia extract(2.5ñ10 µg/mL) is similar to that used in experi-ments by other authors (34, 35) and concentrationsof tested extract in our work (2.5ñ10 µg/mL) can beachieved in plasma during supplementation with thiscommercial extract (35). However, the protectiveaction of the aronia extract on Fg during hyperho-mocysteinemia is still unclear. It should be under-lined that modifications of many of the plasma pro-teins, including Fg, induced by Hcy and its deriva-tives, may stimulate various functional changes(Fig. 1). Because our earlier experiments showedthat different antioxidants (resveratrol, grape seedsextract and aronia extract) protected plasma andplatelet proteins against modifications induced byhyperhomocysteinemia (28-32), now we suggestthat the same protective mechanisms caused by aro-nia extract may exist in our present study, when weused Hcy- and HTL-Fg in the purified system.Moreover, our previous results indicate that aroniaextracts decreased the biotoxic action of Hcy andHTL on the other hemostatic properties of Fg andplasma (i.e., clot formation and fibrin lysis) (37).Results of Arts et al. (38) reported that tea polyphe-nols, including flavonoids react with thiols and ñSHgroups of proteins. We may also suggest that theinteraction between the thiol group of Hcy andpolyphenols may play an important function in theprotective action of aronia extract on Fg ñ treatedwith Hcy or its thiolactone. Therefore, aronia extractseems to be a promising dietary supplement to pre-vent the cardiovascular or the vascular system dur-ing hyperhomocysteinemia.

Acknowledgments

This work was supported by grant 506/1136from University of Lodz, and statutory activities ofInstitute of Soil Science and Plant Cultivation, Pu≥awy(Poland). Special thanks go to J. Maciejewska, J.Malinowska and K. Makaruk (Department of GeneralBiochemistry, University of Lodz).

Statement of interest

None to declare.

Commercial extract from aronia as a modulator... 1613

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Received: 22. 01. 2016


Burn wound infection is one of the most com-mon complications and remains a major publichealth issue affecting all ages groups in both devel-oped and developing countries (1-3). There are threestages of the process of wound healing: inflamma-tion, proliferation and remodeling. The proliferativephase is characterized by angiogenesis, collagen dep-osition, granulation tissue formation, epithelizationand wound contraction. Alterations in any of thesesteps can lead to healing delay or even the inabilityto heal completely (4). New methods of treatingwounds include debridement, irrigation, antibiotics,etc., which possess major drawbacks and unwantedside effects (5). There are many topical agents

applied for burn wounds treatment. Silver sulfadi-azine (SSD) is applied as standard topical therapy inhealing of burn wounds with antibacterial activities(6, 7). Some authors reported that, to delay thewound-healing process (8) and some serious cyto-toxic activities on the host cells (9). Although SSDhas been the standard treatment of burn wound inhospital, alternative treatment for burnt wound that ischeaper and locally available is still needed (10).

The use of natural products to support thewound healing is a common practice in the world.Medicinal plants are used for years in different coun-tries for wound healing, and are preferred because oftheir low toxicity and availability (11-15).

WOUND HEALING ACTIVITY OF EXTRACT FROM THYMUS DAENENSIS INBURN WOUND MODEL: AN EXPERIMENTAL ANIMAL STUDY

SIMIN BABAEIZADEH1, SAEED HEYDARNEJHAD2, ABDOLLAH GHASEMI PIRBALOUTI1,3*,FAHAM KHAMESIPOOR4, ELHAM MOGHTADAEI-KHORASGANI 1

and PARISA HEYDARI-SOURESHJANI 4

1 Department of Medicinal Plants, Shahrekord Branch, Islamic Azad University, P.O. Box 166, Shahrekord, Iran

2 Department of Biology, Shahrekord University, P.O. Box 115, Shahrekord, Iran3 Medicinal Plants Program, College of Natural Science, Massachusetts University,

Amherst, 01003, MA, USA4 Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University,

P.O. Box 166, Shahrekord, Iran

Abstract: Burn wound is one of the most common complications and remains a major public health issueaffecting all ages groups in both developed and developing countries. This study was aimed to evaluate theextract from Thymus daenensis and silver sulfadiazine on healing burn wounds in mice. In this experimentalstudy, the ethanol extract from the aerial parts of T. daenensis (Lamiaceae) was used. Second-degree burnwounds were induced in three groups of eight Balb/C mice each. Group-I: the animals were treated with sim-ple cream (control), Group-II: the animals were treated with simple cream containing the herb extract, andGroup-III: the animals received the standard drug (silver sulfadiazine). The experimental groups were evaluat-ed based on wound area, epithelialization time and histopathological characteristics. There were significant dif-ferences in surface area and the period of burn wound healing between the groups, particularly among Group-II when the animals received the extract of T. daenensis in comparison with control. At the 18th day, there wasno significant improvement in healing percentage of the herb treated (94.6%) in comparison to the animalsreceiving the standard drug (95.8%). The best results of histopathological investigation were obtained with theextract of T. daenensis, when compared to the other group as well as to the control and standard drug. The herbalcream experimentally and histopathologically revealed a burn wound healing activity probably due to theantioxidant and anti-inflammatory activity of its phytochemical contents, especially phenolic compounds.Therefore, T. daenensis accelerated wound healing in mice and thus supports its traditional use.

Keywords: antioxidant activity, burn wound, herbal drug, sulfadiazine, Thymus daenensis, total phenolic

1615

* Corresponding author: e-mail: [email protected] or [email protected]

1616 SIMIN BABAEIZADEH et al.

Thymus L. genus belonging to the Lamiaceaefamily has about 215 species in the world. Thymusspecies grow wild in many regions of Iran. T. daenen-sis Celak subsp. daenensis is a main species of thisgenus in Iran (16). In Iranian traditional medicine, theaerial parts of Thymus species are used as tonic, carmi-native, digestive, antispasmodic, anti-inflammatoryand expectorant (16, 17). T. daenensis essential oil andextracts contain monoterpenes, sesquiterpenes, pheno-lic compounds and flavonoids (18). The essential oiland extracts from the aerial of T. daenensis have beenshown to have antibacterial (18), antifungal (19),antioxidant (20, 21), insecticide (22), anti-adenovirus(23) and immune-modulatory effects (24).

Regarding to the importance of burns and lackof efficient drugs for burn healing in modern medi-cine, research for finding new drugs, especially withnatural origin, is necessary. Therefore, the presentstudy was aimed to evaluate the extract based-cream

of the ethanol from Thymus daenensis versus silversulfadiazine on healing burn wounds in mice(Balb/C). Moreover, antioxidant activity and totalphenolic content were performed to declare the prob-able burn wound healing mechanism of the herb.


Plant material

The aerial parts of T. daenensis (Fig. 1) werecollected from natural habitat of Chahar Mahal vaBakhtiari province, Southwestern Iran (latitude 31O

N; longitude 50O E; altitude 2250 m above sealevel). T. daenensis was identified by regional florasand authors with floristic and taxonomic references(25) and voucher specimen was deposited at theHerbarium of Islamic Azad University ofShahrekord, Iran. The aerial parts of herb wereshade dried and ground into a powder.

Figure 1. Thymus daenensis

Wound healing activity of extract from Thymus daenensis in... 1617

Preparation of extract

The aerial parts of T. daenensis (100 g) wereextracted in Soxhlet apparatus using methanol as sol-vent at 40-50OC for 10 h. The filtered extract was evap-orated using rotary evaporator under reduced pressure.Then, the extract was dissolved in distilled water.

Determination of total phenolic content (TPC)

The TPC of the extract was determined usingthe FolinñCiocalteu method (26). Briefly, 0.5 mL ofthe extract were mixed with 2.5 mL of Folin-Ciocalteuís phenol reagent (5 min at 37OC). Then, 2mL of saturated Na2CO3 (7.5%) solution was added,and the mixture was brought to 10 mL with the addi-tion of deionized distilled water. The mixture wasmaintained at room temperature for 120 min in thedark and then the absorbance of the mixture wasmeasured at 765 nm against a reagent blank using aUV-Vis spectrophotometer (Shimadzu Corp.,Japan). Gallic acid equivalent (GAE) was used as thereference standard and TPC was expressed as mg ofGA equivalents per gram of extract on dry basis.

DPPH radical scavenging activity

The antioxidant activity of the extract wasdetermined using the DPPH assay (27). The sam-ples of the extract were mixed with an equal volumeof 0.2 mM methanol solution of DPPH. The disap-pearance of DPPH was followed spectrophotomet-rically at 515 nm at room temperature. Theabsorbance of the DPPH radical without antioxi-dant against a control was measured daily. Theamount of the sample necessary to decrease theabsorbance of DPPH by 50% (IC50) was calculatedgraphically. The percentage inhibition was calculat-ed according to equation 1:

AC(0) ñ AA(t)% inhibition = [ ññññññññññññ ] × 100 (1)AC(0)

where AC(0) is the absorbance of the control at t = 0min; and AA(t) is the absorbance of the antioxidant(time = 1, 5, 15, and 30 min).

Preparation and maintenance of animals

In this study, we used 40 Balb/C aged 2-3months with the average body weight of 30 ± 2 g. Theanimals were provided from laboratory animal breed-ing and maintenance center. The mice were random-ly allocated into three groups each with eight mice.The animals were considered identical living with12/12 h light/dark cycle at 23 ± 2OC. Food and waterwere provided ad libitum. After one week, study wasinitiated. All procedures and works on animals wereconducted according to Animal Rights MonitoringCommittee of Pharmaceutical Research Center.

Burn induction

Animals were anesthetized with 1.5 mg/kg i.p.of ketamin and xylazine and their dorsal surface wasshaved (disinfected with ethanol) with a sterileblade. The burns were created by Shanmuga et al.(28) method. A cylindrical metal rod with 15 mmdiameter was heated (85-90OC) and pressed to theshaved and disinfected surface for 20 s in miceunder ketamin and xylazine anesthesia.

Grouping of animals

Burns were induced in miceís divided intothree groups as follows: Group I: the animals were treated with simple cream(control), Group II: the animals were treated withsimple cream containing the extract of T. daenensis,and Group III: the animals received the standarddrug (silver sulfadiazine).

Measurement of wound area

The progressive changes in wound area weremeasured in cm2 by tracing the wound boundarieson a transparent paper on every day interval (in total21 days). The burn wound area was calculated usingAuto CAD RL 14 software.

Evaluation of histopathology

For histological examination, the wound areawas removed from the surviving animals at days 6,12 and 18. The skin biopsies were fixed in 10%formaldehyde solution for 48 h and were embeddedin paraffin wax. A 6 µm thickness sections werestained with hematoxylin-eosin stain and observedfor the histopathological changes under light micro-scope (Olympus BX51). Inflammatory cell (neu-trophil), reepithelization, angiogenesis, fibroblasts,vascularization, extracellular matrix, vascularizationand organization of the collagen were qualitativelyevaluated by grading as (-), (+), (++), (+++) (29).

Analysis of data

Results were expressed as the mean ± SEM.The data were analyzed by one way ANOVA andthe means were comprised using Duncanís multiplerange test (DMRT) at p < 0.05 by the MSTAT-C sta-tistical package.


TPC and antioxidant activity

Phenolic compounds are believed to accountfor a major portion of the antioxidant capacity inmany plants. Total phenolic content of the extract ofT. daenensis was 209 mg GAE/g. Results of antiox-


Table 1. Wound burn area (cm2) under different groups in mice (Balb/C).

ANOVAStd. Error Std. Deviation

Mean GroupsDays after

(±SE) (±SD) burn induction

p > 0.05 0.003 0.008 100.012 a Control Day-1

0.001 0.005 100.014 a Silver cream

0.002 0.008 100.011 a T. daenensis

p ≤ 0.01 0.003 0.008 100.012 b Control Day-2

0.182 0.575 99.327 ab Silver cream

0.104 0.331 99.202 a T. daenensis

p ≤ 0.01 0.240 0.536 99.444 b Control Day-3

0.332 1.051 96.986 a Silver cream

0.183 0.580 96.796 a T. daenensis

p ≤ 0.01 0.276 0.617 97.652 b Control Day-4

0.466 1.476 92.520 a Silver cream

0.360 1.139 92.473 a T. daenensis

p ≤ 0.01 0.186 0.417 96.326 b Control Day-5

0.317 1.002 89.058 a Silver cream

0.373 1.182 89.348 a T. daenensis

p ≤ 0.01 0.293 0.656 95.848 b Control Day-6

0.244 0.774 87.100 a Silver cream

0.274 0.867 86.914 a T. daenensis

p ≤ 0.01 0.403 0.698 88.073 b Control Day-7

0.480 1.177 74.181 a Silver cream

3.334 8.822 79.288 a T. daenensis

p ≤ 0.01 0.187 0.324 85.893 b Control Day-8

0.432 1.059 62.093 a Silver cream

3.906 10.334 73.510 ab T. daenensis

p ≤ 0.05 1.131 1.959 77.756 b Control Day-9

1.268 3.107 54.208 a Silver cream

3.274 8.664 65.327 ab T. daenensis

p ≤ 0.01 0.930 1.611 66.556 b Control Day-10

0.622 1.523 41.7367 a Silver cream

3.144 8.320 65.210 b T. daenensis

p ≤ 0.01 0.176 0.305 47.740 b Control Day-11

0.358 0.876 32.326 a Silver cream

1.918 5.076 40.567 ab T. daenensis

p ≤ 0.01 0.250 0.434 40.336 b Control Day-12

1.462 3.581 24.636 a Silver cream

1.653 4.374 31.134 a T. daenensis

p ≤ 0.01 0.125 0.403 39.034 b Control Day-13

0.760 1.078 22.090 a Silver cream

0.987 3.089 29.091 a T. daenensis


idant activity of the extract from T. daenensis indi-cated that the IC50 value was 43.1 mg/mL.

Rate of wound healing

Skin wounds were measured on different days(at days 1 to 18) using DMRT shown in Table 1. Forexample, in Table 1 has shown that the average areaof wound on the 2nd day was 100.012 ± 0.008,99.327 ± 0.575, and 99.202 ± 0.331 cm2 in control,silver cream and T. daenensis extract cream, respec-tively (p < 0.01). The wound area was not signifi-cantly different between silver cream and T. daenen-sis cream on most days of post-burn injury (Table1). In general, wound areas continued to decrease tillthe last day of experiment (day 18).

Histopathological study

In histopathological study, the extent of degreeof inflammation, angiogenesis, hemorrhage, colla-gen fibers and reepithelization were determined(Table 2, Figs. 1-3). The results showed that regard-ing the inflammation, there was a decreasingprocess after days 12 and 18 without any significantdifference between groups on day 6 and 12 but onday 18, the difference was statistically significantbetween T. daenensis extract and silver cream treat-ed groups (Table 2, Figs. 1-3). The collagen fiberorganization in T. daenensis extract cream group

was better than silver cream treated groups on 18th

day (Table 2, Fig. 3). The score of collagen fiberorganization in T. daenensis and silver cream groupsshowed a moderate wound healing score. On the 18th

day, reepithelization was more prominent in T. dae-nensis extract treated group in comparison to silvercream treated group (Table 2, Fig. 3). On day 18th,silver cream treated group appears not to have sig-nificant difference with non-treated control regard-ing reepithelization (Table 2, Fig. 3). No edema orhemorrhage was seen in mice treated with T. dae-nensis and silver creams during the study.

The present study examined the healing effectof T. daenensis extract on burn wounds in mice.Wound healing is a complex process that involvesinflammation, reepithelization, angiogenesis, granu-lation tissue formation, and deposition of interstitialmatrix, beside other events carried out by differenttypes of cells, such as keratinocytes, fibroblasts,inflammatory cells and endothelial cells (4). Resultsof this study indicated that no significant differencewas noticed regarding the wound size between silvercream, and T. daenensis extract cream treatedgroups on days after burn injury. This study demon-strated that the extract of T. daenensis was able toincrease the rate of wound healing for burn wound.The healing effect of T. daenensis might be due toseveral mechanisms such as increasing rate of reep-

Table 1. Cont.

ANOVAStd. Error Std. Deviation

Mean GroupsDays after

(±SE) (±SD) burn induction

p ≤ 0.01 0.395 0.558 32.605 b Control Day-14

0.659 1.141 16.803 a Silver cream

0.050 0.070 24.850 a T. daenensis

p ≤ 0.01 0.060 0.084 28.940 b Control Day-15

0.846 1.466 13.036 a Silver cream

0.215 0.304 20.565 a T. daenensis

p ≤ 0.01 0.515 0.728 24.515 b Control Day-16

0.225 0.389 11.186 a Silver cream

0.065 0.0919 16.265 a T. daenensis

p ≤ 0.01 0.585 0.827 21.585 b Control Day-17

0.185 0.320 8.370 a Silver cream

0.065 0.091 11.935 a T. daenensis

p ≤ 0.01 0.065 0.091 18.065 b Control Day-18

0.078 0.135 4.253 a Silver cream

0.810 1.145 5.390 a T. daenensis

*Means with different letter in a day (after burn induction) are statistically significant at 5% level probability, according to Duncan's mul-tiple range test.


ithelization and neovascularization, scavenging ofdestructive free radicals, inflammation reductionand control of infection by the effects of antioxidant,anti-inflammatory and antimicrobial constituents ofthe plants used in the cream, especially polyphenolsand tannins as the main components. Silver sulfadi-azine cream is the most common topical product

used for treatment of burn injuries. The antimicro-bial efficacy of this agent is probably the main rea-son for its widespread use in burn wounds (30).However, delayed wound healing following treat-ment is the most important clinical adverse effect ofsilver topical agents which limits their long-termuse, especially on broad wounds (7, 31). In the con-

Table 2. Histopathological evaluations of burn wounds in animals after 6, 12, and 18 days of topical application.

Inflammation Treatments Day cells Angiogenesis Hemorrhage Collagen fibers Reepithelization

(Neutrophil)

Control 6 + + - + - -

12 + + - + + -

18 + - + + -

Silver sulfadiazine 6 + - + + +

12 + - - + +

18 + + + ++ -

T. daenensis cream 6 + - - + +

12 + + - ++ +

18 + + ++ ++

+: slight, ++: moderate, +++: extensive, -: absent.

Figure 2. Histological evaluation after six days of burn wound creation in three groups (a, b, c). a: Control (H&E40X). 1: Inflammatorycells; 2: Hemorrhage; 3: Collagen fibers. b: Silver sulfadiazine cream (H&E40X). 1:Inflammatory cells; 2: Hemorrhage; 3: Collagen fibers.c: Thymus daenensis cream (H&E40X). 1: Inflammatory cells; 2: Collagen fibers; 3: Hemorrhage

Figure 3. Histological evaluation after 12 days of burn wound creation in three groups (a, b, c). a: Control (H&E40X). Angiogenesis +inflammatory cells + hemorrhage. b: Silver sulfadiazine cream (H&E40X). 1: Collagen fibers; 2: Inflammatory cells. c: Thymus daenen-sis cream (H&E40X). Angiogenesis + collagen fibers + inflammatory cells

a b c

a b c


trol group, there was a loose collagen matrix withinterstitial edema and hemorrhage. This is becausecollagen plays an essentials role in wound healingand as a principal component of connective tissueprovides a structural framework for the tissue regen-eration (Table 2). On day 18, samples revealed anincrease in collagen fibers organization and reep-ithelization in animals treated with T. daenensis andsilver sulfadiazine creams in comparison to controlgroup (Fig. 4). The reepithelization parameter for T.daenensis treated group was significantly higherthan for controls group (Fig. 4).

T. daenensis has been used in Iranian tradition-al medicine in different parts of Iran. Results ofinvestigations showed that the main compounds inT. daenensis were oxygenated monoterpenes, in par-ticular thymol and carvacrol (32, 33), T. daenensisexhibits significant antimicrobial and antioxidantactivities (21, 34, 35). Ghasemi Pirbalouti et al., (36)also reported that T. daenensis essential oil weaklyinhibited Streptococcus iniae, with MIC = 312µg/mL, while the activity of T. daenensis essentialoil against C. albicans was moderate (37). Theinhibitory effect of T. daenensis on the mycelialgrowth of some pathogenic fungi was compared toother essential oils (38). According to our resultsand another investigations have been performed onthe antioxidant potential of T. daenensis essential oiland extract to underline the traditional uses of theherb against inflammatory diseases (20, 39, 40).Antioxidants such as phenols and flavonoids in theessential oils and extracts from the herbs may helpto control wound oxidative stress and thereby accel-erate wound healing (41, 42). Oxidants are inhibito-ry factors to wound healing due to their cell damageability. The topical application of natural com-pounds with free radical scavenging properties on

patients or animals have shown significant improve-ment in wound healing and protection tissues fromoxidative damage. Antioxidants could also have animportant role in survival of ischemic skin flaps orpromotion of wound healing (43).

CONCLUSIONS

Thymus species are well known as aromaticand medicinal plants because of their biological andpharmacological properties. In this study, we evalu-ated the in vivo properties of Thymus daenensis onburn wound healing in mice. There were significantdifferences in surface area and the period of burnwound healing between the groups, particularlywhen using T. daenensis cream. Nevertheless, therewas no significant differences between the period ofburn wound healing between creams of T. daenensisand sulfadiazine. Reepithelization, collagen fibersand fibrous tissue were more prominent in bothcreams. Therefore, it can be concluded that topical-ly applied T. daenensis may be a suitable substitutefor silver sulfadiazine. Further studies shouldaddress these issues in humans.


There is no conflicts of interest among theauthor who contributed to this study.

REFERENCES

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Figure 4. Histological evaluation after 18 days of burn wound creation in three groups (a, b, c). a: Control (H&E40X). 1: Collagen fibers;2: inflammatory cells. b: Silver sulfadiazine cream (H&E40X). 1: Inflammatory cells + Hemorrhage; 2: Edema; 3: Collagen fibers andfibrous tissue. 4: Epithelization tissue. c: Thymus daenensis cream (H&E40X). 1: Inflammatory cells; 2: Collagen fibers; 3: Epithelizationtissue

a b c


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Received: 4. 04. 2016


The role of antiseptics as an alternative thera-peutic approach to eliminate bacterial strains that areresistant to most systemic antibiotics has gained con-siderable importance since the emergence of multire-sistant bacteria in human and companion animals (1).There is no current evidence for bacterial resistanceto antiseptics as frequently or as quickly and they donot result in allergic reactions as frequently either.

Chlorhexidine, a bisbiguanide antiseptic, hasexcellent efficacy against Gram-positive bacteria,although its efficacy against Gram-negative bacteriais less pronounced (1).

Furthermore, chlorhexidine has shown efficacyagainst many fungi and enveloped viruses.Chlorhexidine is characterized by strong binding to theskin and ability to adsorb to negatively charged surfaces.

The antimicrobial effect of chlorhexidineinvolves the attraction and adsorption of cationicmolecules to the cell surface of microorganisms,promoting change in cell membrane permeability,resulting in the loss of intracellular components andosmotic imbalance in the cell.

Depending on the concentration, all vegetativebacterial forms are quickly and completely

PHARMACEUTICAL TECHNOLOGY

DETERMINATION OF ANTIMICROBIAL ACTIVITY OF CHLORHEXIDINE GEL

LINA BABICKAIT�1, KRISTINA RAMANAUSKIEN�2, AIDAS GRIGONIS1, MARIJA IVA�KIEN�2, GINTARAS DAUNORAS2, IRENA KLIMIEN�3,

MARIUS VIRGAILIS3, GINTARAS ZAMOKAS1, ASTA MARIJA INK�NIEN�2* and ALGIMANTAS PETRAS MATUSEVI»IUS1

1Lithuanian University of Health Science, Veterinary Academy, Faculty of Veterinary Medicine, Department of Non-Infectious Diseases,

Til��s St. 18, 47181 Kaunas, Lithuania2Lithuanian University of Health Science, Medical Academy,

Faculty of Pharmacy, Department of Clinical Pharmacy,Sukil�� av. 13, 50009 Kaunas, Lithuania

3Lithuanian University of Health Science, Veterinary Academy, Faculty of Veterinary Medicine, Institute of Microbiology and Virology,

Til��s St. 18, 47181 Kaunas, Lithuania

Abstract: Many researchers have advocated in recent times that antiseptic use in healing wounds should be dis-couraged. Antiseptics have been found to retard healing of wounds. Poloxamer 407 shows thermoreversibleproperties, which are of the utmost interest in optimizing drug formulation (fluid state at room temperaturefacilitating administration and gel state above sol-gel transition temperature, at body temperature, promotingprolonged release of pharmacological agents). Chlorhexidine, a commonly used antiseptic, is known to be lesstoxic on granulation cells. Acting as an antiseptic, it is an effective bactericidal agent against the most categoriesof microbes, including bacteria, yeast, and viruses. Objective of this study was to evaluate antimicrobial activ-ity of chlorhexidine containing poloxamer gel to Gram-positive and Gram-negative bacteria in vitro.Chlorhexidine gels and chlorhexidine aqueous solutions have different antibacterial activity to S. aureus, E. fae-calis, E. coli and P. aeruginosa strains in vitro. It depends on concentration and dosage form of antiseptic. Studyresults confirmed that antimicrobial activity of gel depends on active ingredient concentration in antiseptic. Thebest inhibition effect for both of reference and wild-type bacteria was obtained for 1% chlorhexidine gel.Summarizing the results and assessing the characteristics of the gel ingredients, it can be suggested usingchlorhexidine gels in veterinary medicine.

Keywords: poloxamer 407, chlorhexidine, antiseptic, gel

1623


1624 LINA BABICKAIT��

destroyed due to contact with antiseptic or it inhibitstheir growth. Chlorhexidine does not allow bacterialspores overgrowth (2). Moreover, chlorhexidine hasantifungal effect and damages the cytoplasm ofyeast. It affects envelope viruses also. Chlorhexidineexerts its major antibacterial effects by interferingwith the function of cellular membranes, the pri-mary site of action being the cytoplasmic mem-brane. Some efficacy studies have demonstrated thatchlorhexidine induces a more significant reductionof bacterial cultures than other antimicrobial com-pounds. Binding of biguanide groups to the mem-brane phospholipids induces structural modifica-tions with leakage of the intracellular componentsand structural modifications of bacterial surface.The bacterial wall membrane alteration and osmoticbalance are changed due to lipophilic groups of anti-septic. Chlorhexidine inhibits activity of enzymesand disturbs potassium ions, nucleotides and aminoacids transport through cell membrane (3).

Poloxamer has been developed as local anes-thetic gel for sustained pain relief and as deliverysystem for ophthalmic drugs. It is low toxic,mucomimetic (4).

Poloxamer has a tri-block structure: polyethyl-ene oxide-polypropylene oxide-polyethylene oxide.They are used in pharmaceutical formulations as sur-factants, emulsifying agents, solubilizing agents, dis-persing agents, and in vivo absorbance enhancers (5).

Poloxamers have gained increasing attentiondue to their abilities in repairing biological mem-branes damaged by trauma and diseases (6, 7).

After topical application, poloxamer dries anda thin protective film consists as it warms to bodytemperature. This film prevents the active ingredi-ents evaporation and leakage from application area,protects skin from mechanical enviroment factors.Poloxamer gel absorbs sweat gland secretions,which may be irritating under long-term occlusion(8). Local tolerance was described as very satisfac-tory even in the treatment of thermal burns (6).

The gel not only does provide a non-toxicdetergent covering to the wound, but specific stud-ies suggest that the gel itself may have a beneficialaction, accelerating wound healing. This makespoloxamer a very suitable vehicle for gels intendedto be applied for ulcers and traumatic lesions (5).

Studies have shown that poloxamer hasimmunomodulatory properties and reduced the neu-trophils activation (5, 6).

This chemical substance is attractive, because itis transformed from low-viscosity solution at low tem-perature to a semisolid gel at higher temperature (5).Poloxamer gel shows temperature-dependent gelation(gel consistency at 37OC and liquid consistency at4OC) (9). In spite of pharmaceutical properties, thethermoreversible nature of poloxamer is of the utmostinterest in optimizing drug formulation, which pro-mote the prolonged release of pharmacological agents(5). The thermogelation results from interactionsbetween different segments of the copolymer. As tem-perature increases, poloxamer copolymer moleculesaggregate into micelles. This micellization is due tothe dehydration of hydrophobic blocks (6).

EXPERIMENTAL

Composition

Investigated antiseptic gel was composed ofchlorhexidine, poloxamer 407 and water (Table 1).Chlorhexidine digluconate aqueous solution (20%)was added into purified water. Then, poloxamer 407(polyoxyethylene-polyoxypropylene-polyoxyethyl-ene polymer) was added and slowly mixed.Compound was kept in refrigerator at temperature+4OC until dissolved and formed hydrogels when itwas kept in room temperature.

pH Measurement

The pH of chlorhexidine gel was determinedby using a HD 2105.1 pH-meter (Delta OHM, Italy).A 5% solution was produced for the pH assessment.

Table 1. Composition of chlorhexidine digluconate gels (%).

Hydrogel ingredients CHX-gel-0.25 CHX-gel-0.5 CHX-gel-1

Purified water 73.75 72.5 70

20% chlorhexidine digluconate aqueous solution

1.25 2.5 5

Poloxamer 407 25 25 25

Note: CHX-gel-0.25 = 0.25% chlorhexidine-poloxamer gel; CHX-gel-0.5 = 0.5% chlorhexidine-poloxamer gel;CHX-gel-1 = 1% chlorhexidine-poloxamer gel.

Determination of antimicrobial activity of chlorhexidine gel 1625

An appropriate amount of gel was mixed with puri-fied water and stirred on a magnetic stirrer IKAM-AGÆ C-MAG HS7 (IKA-Werke GmbH & Co.KG,Staufen, Germany), for 30 min at +50OC tempera-ture. Then, the solution was cooled and filteredthrough a paper filter with 20-25 µm pores (DP 411,AlbetÆ Filtration & Separation Technology, Spain).Two and a half grams gel was placed in a glass with50 mL of purified water and stirred with a glass roduntil the gel was completely dissolved. The elec-trodes were rinsed with purified water after eachmeasurement (n = 3) using the pH-meter pH/mVmeter Delta OHM HD 2105.1 (Delta OHM, Italy).Results are shown as the arithmetic average.

Antimicrobial activity test

Antimicrobial efficacy of antiseptic gel was car-ried in vitro agar by diffusion method using Mueller-Hinton agar (Mueller-Hinton agar (CM 0337) OxoidLTD, Basingstoke, Hampshire, England).

Four standard Gram-positive and Gram-nega-tive microorganisms: Staphylococcus aureus(ATCC 25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC 25922),Pseudomonas aeruginosa (ATCC 27853) and fourwild strains of Staphylococcus aureus, Enterococcusfaecalis, Escherichia coli and Pseudomonas aerugi-nosa were used for this study in vitro.

Different concentrations (0.25, 0.5 and 1%) ofchlorhexidine-poloxamer gel were used.

According to the standard approved by theClinical and Laboratory Standards Institute (CLSI),35 mL of liquid Mueller Hinton agar was pouredinto each Petri plate (10 cm in diameter) and left ina horizontal position to clot. Inoculum was stan-dardized to 0.5 McFarland turbidity. Inoculationwith each bacteria strain was performed by usingsterile swabs brushed across the media. Six roundwells, 7 mm in diameter, were punched in the culti-vated agar plate with a sterile copper tube.

Figure 1. The antibacterial activity of chlorhexidine-poloxamer gel and chlorhexidine aqueous solution on S. aureus (CHX-gel-0.25 =0.25% chlorhexidineñpoloxamer gel, CHX-gel-0.5 = 0.5% chlorhexidineñpoloxamer gel, CHX-gel-1 = 1% chlorhexidine-poloxamer gel,CHX-W-0.25 = 0.25% chlorhexidine aqueous solution, CHX-W-0.5 = 0.5% chlorhexidine aqueous solution, CHX-W-1 = 1% chlorhexi-dine aqueous solution, POL = poloxamer)


Chlorhexidine gel, poloxamer gel and chlorhex-idine aqueous solution (the control) were filled inwells (0.1 mL in each well). Two wells in one Petriplate were given for each test material. Fifteen Petriplates were intended for each bacterial strain (fivePetri plates for each test substance concentration).Petri plates were incubated for 24 h at 36OC.

Chlorhexidine gel antibacterial activity wasassessed in vitro after 24 h incubation. The zones ofinhibition were measured using millimeter scale,passing through its diameter. If zone of inhibitionwas not present around the well, it was consideredthat the investigated material does not has a bacteri-cidal effect on the tested bacteria strains.

RESULTS

The quality of tested antiseptic gels was inves-tigated by analyzing their pH value and antimicro-bial activity. pH of all tested gels were approximate-ly 6.5.

Chlorhexidine gel (0.25% concentration -CHX-gel-0.25) most suppressed growth of reference

strain of S. aureus (diameter of inhibition zone: 15.7± 0.15 mm, p < 0.05) and reference strain of E. coli(diameter of inhibition zone: 15.3 ± 0.15 mm, p <0.05). Less activity was observed on other bacterialstrains: wild-type strain of S. aureus was 1.9-4.5%less resistant (diameter of inhibition zone: 15.0 ±0.21 mm), wild-type strain of E. faecalis - up to 26%(diameter of inhibition zone: 11.6 ± 0.16 mm), ref-erence strain of E. faecalis - 6.5-8.9% (diameter ofinhibition zone: 14.3 ± 0.15 mm), wild-type strain ofE. coli - 12.4-14.6% (diameter of inhibition zone:13.4 ± 0.3 mm), wild-type strain of P. aeruginosa -43.1-44.6% (diameter of inhibition zone: 8.7 ± 0.33mm), reference strain of P. aeruginosa - up to 29.3%(diameter of inhibition zone: 11.1 ± 0.1 mm). Theweakest inhibitory activity was seen on referenceand wild-type strains of P. aeruginosa (with thediameter of the inhibition zone reaching 1.1 ± 0.1mm and 8.7 ± 0.33 mm, respectively) (p < 0.05)(Figs. 1-4). Inhibition effect of 0.25% chlorhexidinegel compared to the aqueous 0.25% chlorhexidinesolution for all bacterial strains was significantlyweaker: wild-type strain of S. aureus by 33.9%

Figure 2. The antibacterial activity of chlorhexidine-poloxamer gel and chlorhexidine aqueous solution on E. faecalis


(inhibition zones diameters 15.0 ± 0.21 mm and22.7 ± 0.15 mm, respectively, p < 0.05), referencestrain by 30.5% (inhibition zones diameters 15.7 ±0.15 mm and 22.6 ± 0.16 mm, respectively, p <0.05), E. faecalis wild-type strain by 31.0% (inhibi-tion zones diameters 11.6 ± 0.16 mm and 16.8 ±0.13 mm, respectively, p < 0.05), reference strain by36.2% (inhibition zones diameters 14.3 ± 0.15 mmand 22.4 ± 0.16 mm, respectively, p < 0.05), E. coliwild-type strain by 25.6% (inhibition zones diame-ters 13.4 ± 0.3 mm and 18.0 ± 0.15 mm, respective-ly, p < 0.05), reference strain by 26.8% (inhibitionzones diameters of 15.3 ± 0.15 mm and 20.9 ± 0.23mm, respectively, p < 0.05), P. aeruginosa wild-typestrain by 53.2% (inhibition zones diameters of 8.7 ±0.33 mm and 18.6 ± 0.16 mm, respectively, p <0.05), reference strain by 35.8% (inhibition zonesdiameters of 11.1 ± 0.1 mm and 17.3 ± 0.15 mm,respectively, p < 0.05) (Figs. 1-4).

The 0.5% concentration chlorhexidine gel(CHX-gel-0.5) most suppressed growth of referencestrain of S. aureus (diameter of inhibition zone: 17.2± 0.2 mm, p < 0.05) and reference strain of E. fae-

calis (diameter of inhibition zone: 17.0 ± 0.15 mm,p < 0.05). Less activity was obtained on other bacte-rial strains: wild-type strain of S. aureus was affect-ed 2.3% less (diameter of inhibition zone: 16.8 ± 0.2mm), wild-type strain of E. faecalis - up to 27.9%(diameter of inhibition zone: 12.4 ± 0.16 mm), wild-type strain of E. coli - up to 14.5% (diameter of inhi-bition zone: 14.7 ± 0.3 mm), reference strain of E.coli - up to 8.7% (diameter of inhibition zone: 15.7± 0.15 mm), wild-type strain of P. aeruginosa - up to34.3% (diameter of inhibition zone: 11.3 ± 0.26mm), reference strain of P. aeruginosa - up to 27.3%(diameter of inhibition zone: 12.5 ± 0.17 mm). Theweakest inhibitory activity was seen on wild-typestrains of E. faecalis (diameter of the inhibition zone1.1 ± 0.16 mm) and P. aeruginosa (inhibition zonediameter 11.3 ± 0.26 mm) (p < 0.05) (Figs. 1-4).Inhibition effect of 0.5% chlorhexidine gel com-pared to the aqueous 0.5% chlorhexidine solutionfor all bacterial strains was significantly weaker:wild-type strain of S. aureus by 29.7% (diameters ofinhibition zones 16.8 ± 0.2 mm and 23.9 ± 0.18 mm,respectively, p < 0.05), reference strain by 25.9%

Figure 3. The antibacterial activity of chlorhexidine-poloxamer gel and chlorhexidine aqueous solution on E. coli


(diameters of inhibition zones 17.2 ± 0.2 mm and23.2 ± 0.25 mm, respectively, p < 0.05), E. faecaliswild-type strain by 30.7% (diameters of inhibitionzones 12.4 ± 0.16 mm and 17.9 ± 0.18 mm, respec-tively, p < 0.05), reference strain by 33.1% (diame-ters of inhibition zones 17.0 ± 0.15 mm and 25.4 ±0.16 mm, respectively, p < 0.05), E. coli wild-typestrain by 23.8% (diameters of inhibition zones 14.7± 0.3 mm and 19.3 ± 0.26 mm, respectively, p <0.05), reference strain by 29.9% (diameters of inhi-bition zones 15.7 ± 0.15 mm and 22.4 ± 0.22 mm,respectively, p < 0.05), P. aeruginosa wild-typestrain by 43.5% (diameters of inhibition zones 11.3± 0.26 mm and 20.0 ± 0.47 mm, respectively, p <0.05), reference strain by 34.9% (diameters of inhi-bition zones 12.5 ± 0.17 mm and 19.2 ± 0.13 mm,respectively, p < 0.05) (Figs. 1-4).

The 1% concentration chlorhexidine gel (CHX-gel-1) most suppressed growth of reference strain ofE. faecalis (diameter of inhibition zone: 19.5 ± 0.17mm, p < 0.05) and wild-type strain of S. aureus(diameter of inhibition zone: 19.3 ± 0.21 mm, p <0.05). Less activity was obtained on other bacterialstrains: reference strain of S. aureus by up to 3.1%

(diameter of inhibition zone: 18.9 ± 0.31 mm), wild-type strain of E. faecalis by up to 26.1% (diameter ofinhibition zone: 14.4 ± 0.16 mm), wild-type strain ofE. coli by up to 16.9% (diameter of inhibition zone:16.2 ± 0.13 mm), reference strain of E. coli by up to8.7% (diameter of inhibition zone: 17.8 ± 0.2 mm),wild-type strain of P. aeruginosa by up to 23.1%(diameter of inhibition zone: 15.0 ± 0.15 mm), refer-ence strain of P. aeruginosa by up to 21.0% (diame-ter of inhibition zone: 15.4 ± 0.16 mm). The weakestinhibitory activity was seen on wild-type strains of E.faecalis (diameter of inhibition zone reaching 14.4 ±0.16 mm) and P. aeruginosa (diameter of inhibitionzone 15.0 ± 0.15 mm) (p < 0.05) (Figs. 1-4).Inhibition effect of 1% chlorhexidine-poloxamer gelcompared to the aqueous 1% chlorhexidine solutionfor all bacterial strains was weaker: wild-type strainof S. aureus by 19.9% (diameters of inhibition zones19.3 ± 0.21 mm and 24.1 ± 0.1 mm, respectively, p< 0.05), reference strain by 21.9% (diameters of inhi-bition zones 18.9 ± 0.31 mm and 24.2 ± 0.13 mm,respectively, p < 0.05), E. faecalis wild-type strainby 23.8% (diameters of inhibition zones 14.4 ± 0.16mm and 18.9 ± 0.23 mm, respectively, p < 0.05), ref-

Figure 4. The antibacterial activity of chlorhexidine-poloxamer gel and chlorhexidine aqueous solution on P. aeruginosa


erence strain by 21.4% (diameters of inhibition zones19.5 ± 0.17 mm and 24.8 ± 0.13 mm, respectively, p< 0.05), E. coli wild-type strain by 20.6% (diametersof inhibition zones 16.2 ± 0.13 mm and 20.4 ± 0.16mm, respectively, p < 0.05), reference strain by24.3% (diameters of inhibition zones 17.8 ± 0.2 mmand 23.5 ± 0.17 mm, respectively, p < 0.05), P.aeruginosa wild-type strain by 26.5% (diameters ofinhibition zones 15.0 ± 0.15 mm and 20.4 ± 0.16mm, respectively, p < 0.05), reference strain by24.5% (diameters of inhibition zones 15.4 ± 0.16mm and 20.4 ± 0.16 mm, respectively, p < 0.05)(Figs. 1-4).

DISCUSSION

Differences in cutaneous pH among species aredescribed and it have ranged from 5.5 (for a guineapig) to 8.1 (for a sheep) (10). Thus, chlorhexidinegels are suitable for skin application and can be usedto treat wounds. Gels form a protective film whichprotects the wound from environmental factors andhave antibacterial activity. Moreover, poloxameritself positively effects and accelerates ulcers andtraumatic wounds healing (10-12).

The viscosity results correspond to data pre-sented in the literature: poloxamer 407 (polyoxyeth-ylene-polyoxypropylene-polyoxyethylene copoly-mer) system has thermosensitive property that thesystem is able to change from solution stage at lowtemperature to the viscous gel at high temperature(11). The study has shown that the viscosity of thegel does not have any influence on the release of theactive ingredient from hydrophilic matrices.

Chlorhexidine gels and chlorhexidine aqueoussolutions have different antibacterial activity to S.aureus, E. faecalis, E. coli and P. aeruginosa strainsin vitro (Figs. 1-4). It depends on concentration anddosage form of antiseptic. Poloxamer alone did notaffect to investigate bacteria growth and inhibitionzones around the wells were not present in Petriplates. Inhibition zones were smaller in diameterwhen chlorhexidine gel was used compared withchlorhexidine aqueous solutions. As the chlorhexi-dine concentration in the gel or aqueous solutionincreased, the inhibition zones around the wellsincreased in diameter as well.

The results of this study and the data presentedin scientific literature confirm that chlorhexidine-poloxamer gel has a broad-spectrum antimicrobialeffect (14). Specifically for a broad antimicrobialactivity and low toxicity, chlorhexidine is widelyused not only in human medicine, but in veterinarymedicine too.

Shampoos containing chlorhexidine, becauseof it antimicrobial efficacy, antifungal and antiviraleffect are used in veterinary dermatology.Moreover, chlorhexidine does not affect corneo-cytes. In comparison with other antiseptics,chlorhexidine is considered to be more effective andless skin irritating. It does not inhibit platelets,macrophages, leukocytes, fibroblasts migration tothe wound, so chlorhexidine stimulates open woundhealing and reduces risk of recurrent infection.

It has been determined that more bacteria areisolated from the distal hair shaft than from skin sur-face, and chlorhexidine inhibits bacterial growth onhair shaft for at least 7 days. While other antiseptics(ethyl lactate, benzoyl peroxide) act on skin surfacebetter (15).

Chlorhexidine is also used for cows teats anti-septic. After month of using solutions containingchlorhexidine, the percentage reduction in the newintramammary infection was 50ñ75% forStaphylococcus spp., Streptoccocus spp.,Enterobacteriae spp., Bacillus spp. The overall rateof new intramammary infections by differentmicroorganisms was significantly reduced by66.66%, thereby increasing milk production andimproving the quality of milk (16).

Study results confirmed that antimicrobialactivity of gel depends on active ingredient concen-tration in antiseptic (Figs. 1-4). The best inhibitioneffect for both of reference and wild-type bacteria of1% chlorhexidine gel was obtained. It was observedthat research carried out on both reference and wild-type bacteria are appropriate, because antibacterialactivity of chlorhexidine gels to wild-type bacteriawas less efficient. Probably this rule can be appliedfor other antiseptics as well. This suggests, thatwild-type bacteria strains are more resistant tochlorhexidine than the reference bacteria strains.

Chlorhexidine aqueous solution is more anti-bacterial efficient, because chlorhexidine fasterreleases from aqueous solution compared with gel.Affinity of the cationic chlorhexidine for the nega-tively charged carboxymethylcellulose is expectedand binding of the functional groups of the polymerand the ionic sites of chlorhexidine can result indelayed release of the drug from the polymeric form(17). However, chlorhexidine in gel system is char-acterized by prolonged action and it is important forwounds treatment in clinical therapy.

Poloxamer has adhesive and mucomimeticproperties, and chlorhexidine is characterized byantimicrobial and antifungal effect. Selected jellify-ing material as drug vehicle is of great interest inhuman medicine. In conclusion, antiseptic chlorhex-


idine gels are characterized by antimicrobial activi-ty, positive organoleptic and physicochemical prop-erties. Chlorhexidine and poloxamer goes well toeach other in the gel, complement each other actionand probably act synergistically.

In summary, the results and assessing the char-acteristics of the gel ingredients, it can be suggestedfor using chlorhexidine gels in veterinary medicine.There are planning to do preclinical study ofchlorhexidine gels with laboratory animals and tar-get animals in the future. It is expected, that polox-amer in combination with chlorhexidine will havepositive effect to damaged tissues and stimulatewound healing.

REFERENCES

1. Banovic F., Bozic F., Lemo N.: Vet. Dermatol.24, 409 (2013).

2. Salami A.A., Imosemi I.O., Owoeye O.O.: Int.J. Morphol. 24, 673 (2006).

3. Odore R., Valle V.C., Re G.: Vet. Res.Commun. 24, 229 (2000).

4. Wannachaiyasit S., Phaechamud T.: JOM. 3,165 (2010).

5. Patel H.R., Patel R.P., Patel M.M.: Int. J.PharmTech Res. 2, 299 (2009).

6. Dumortier G., Grossiord J.L., Agnely F.,Chaumeil C.J.A.: Pharm. Res. 12, 2709 (2006).

7. Wu G., Khant H.A., Chiub W., Lee K.Y.C.:RSC. 5, 1496 (2009).

8. Nair V., Panchagnula R.: Pharm. Res. 47, 555(2003).

9. Carceles C.M., Serrano J.M., Marin P.,Escudero E., Fernandez-Varon E.: J. Vet. Med.53, 300 (2006).

10. Matousek J.L., Campbell K.L.: Vet. Dermatol.13, 293 (2002).

11. Beynon T., Laverty D., Baxter A., Forsey P.,Grocott P.: J. Pain Symptom Manag. 26, 776(2003).

12. Paolantonio M., DíAngelo M., Grassi R.F.,Perinetti G., Piccolomini R. et al.: J.Periodontol. 79, 271 (2008).

13. Puig-Silla M., Montiel-Company J.M.,Almerich-Silla J.M.: Med. Oral Patol. Oral Cir.Bucal. 13, E257 (2008).

14. Vianna M.E., Gomes B.P., Berber V.B., ZaiaA.A., Ferraz C.C., Souza-Filho F.J.: Oral Surg.Oral Med. Oral Pathol. Oral Radiol. Endod. 97,79 (2004).

15. Kloos I., Straubinger R.K., Werckenthin C.,Mueller R.S.: Vet. Dermatol. 24, 250 (2013).

16. Sharma S.K., Joshi M., Shukla P.C.: Vet. Pract.7, 55 (2006).

17. Fini A., Bergamante V., Ceschel G.C.: Pharma-ceutics 3, 665 (2011).

Received: 10. 12. 2015


Hypertension is the most common cardiovas-cular disease and one of the major leading causes ofcardiovascular morbidity and mortality in the world,which accounts for 20-50% of all cardiovasculardeaths. It could be defined in its simplest form asuncontrolled and high blood pressure. The mainapproach in its treatment is to perform constantmonitoring and to use daily orally administrateddrugs to achieve blood pressure control (1).Candesertan cilexetil is an angiotension II receptorblocker and classified in the BCS as a class II drug.It is widely used for the treatment of heart failureand hypertension. According to other angiotensionreceptor antagonists, the major superiority of can-desertan cilexetil is its higher potency and lack ofside effects. However, after oral administration, it israpidly hydrolyzed to its active form and its oralbioavailability is limited because of its inadequateabsorption. This low oral bioavailability (approxi-mately 40%) is resulted from its poor aqueous solu-

bility (0.0003 mg/mL). The major problem in thedevelopment of more efficient formulations of can-desertan cilexetil is its poor solubility in physiolog-ically relevant pH conditions and aqueous media (2-4). Therefore, many strategies have been developedby several researchers to solve the solubility prob-lems and to enhance the oral bioavailability of can-desertan cilexetil. Nano-sized drug carriers, espe-cially solid lipid nanoparticles (SLNs) can be themain approach developed for this purpose due totheir ability to enhance the lymphatic transport ofthe lipophilic drugs and to improve the oral bioavail-ability of the active molecules by this way (5-8).

In recent years, SLNs have been of great inter-est and have gained attention because of their uniquecharacteristics. The main advantages of the SLNsare: high storage and biological stability comparedto liposomes, utilization of non-toxic and biocom-patible ingredients, the use of organic solvents beingavoidable in the production process, convenience of

DEVELOPMENT OF SOLID LIPID NANOCARRIERS FOR ORAL DELIVERYOF CANDESERTAN CILEXETIL

TIMUCIN UGURLU1*, AYSUN NALBANTOGLU2 and CEYDA TUBA SENGEL-TURK3

1Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 34668 Haydarpasa, Istanbul, Turkey

2Zentiva Health Products Research and Development Center, 39780 Luleburgaz, Kirklareli, Turkey3Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Technology,

06100 Tandogan, Ankara, Turkey

Abstract: Candesertan cilexetil is a Biopharmaceutics Classification System (BCS) Class II drug possessinghigh permeability but low aqueous solubility; hence its oral bioavailability is limited in terms of the solubilityrate. The aim of this research was to develop solid lipid nanoparticle (SLN) drug delivery systems of can-desertan cilexetil to enhance its aqueous solubility, thereby improving the oral bioavailability of the drug. SLNformulations were produced using a combined technique of high shear homogenization and ultrasonicationmethod. Drug/lipid and surfactant/co-surfactant ratios of the candesertan cilexetil loaded SLNs were investi-gated based on various final characteristics of the nanocarriers; namely, encapsulation efficiency, average par-ticle diameter, size distribution, surface charge, thermal behavior, and in vitro drug release profiles. Lipid basednanocarriers of candesertan cilexetil displayed spherical particles having a nanometer size. High encapsulationefficiencies were obtained due to the high lipid solubility of the drug. DSC analysis demonstrated the transfor-mation of the crystalline structure of candesertan cilexetil to amorphous form into the SLN formulations andthere was no interaction between the drug and the excipients. Consequently, the oral delivery of candesertancilexetil with the design of CompritolÆ 888 ATO based lipid nanocarriers may lead to an increase in bioavail-ability of the drug and thus, more effective therapy may be obtained.

Keywords: candesertan cilexetil, hypertension, SLN, CompritolÆ 888 ATO, PoloxamerÆ 188, oral drug delivery

1631

* Corresponding author: e-mail: [email protected]; phone: (90) 216 418 50 29

1632 TIMUCIN UGURLU et al.

the scalability and adaptability of manufacturingtechnologies to the pharmaceutical industry as com-pared to polymeric nanoparticles (9, 10).

In this research, SLN based nanocarriers ofcandesertan cilexetil were designed and developedas an approach to improve the solubility and therebythe bioavailability of drug. Candesertan cilexetilencapsulated SLN formulations were prepared witha high shear homogenization technique followed byan ultrasonication method. Prepared SLNs wereevaluated with respect to the encapsulation efficien-cy, average particle diameter, particle size distribu-tion (polydispersity index, PDI), surface charge, andin vitro drug release rate. Candesertan cilexetilloaded SLN formulations were previously reportedby some workers (5-7, 11, 12), however the excipi-ents used will firstly be combined with each otherfor preparing the SLN formulation of candesertancilexetil. For these purposes, the interaction of drugexcipients was also deeply investigated in ourresearch.

EXPERIMENTAL

Candesartan cilexetil was obtained fromRanbaxy Laboratories (Gurgaon, Haryana, India).CompritolÆ 888 ATO was supplied by GattefossÈLaboratories (Saint-Priest, France). PoloxamerÆ

188 was purchased from BASF (Ludwigshafen,Germany). Polyethylene sorbitol ester (Tween 80)was obtained from Sigma-Aldrich (St. Louis, MO,USA). All other chemicals used were at least ofreagent grade.

Candesertan cilexetil-incorporated SLN for-mulations were produced using a high shear homog-enization technique followed by an ultrasonicationprocess. The drug/lipid (candesertan cilexetil/CompritolÆ 888 ATO) (1/30, 1/60, 1/120) ratio andsurfactant/co-surfactant (PoloxamerÆ 188/Tween80) ratio (1/0.5, 1/2) were selected as the investigat-ed formulation parameters in this research (Table 1).The effects of these formulation parameters on vari-ous physico-chemical characteristics of the SLNswere evaluated. 1/2 ratio of the surfactant/co-surfac-tant level was investigated solely on the lowest(1/30) and the highest (1/120) ratios of the drug/lipidlevel. For the preparation of the SLN formulations,firstly CompritolÆ 888 ATO was melted at 80OCand candesertan cilexetil was added to this lipidphase. The lipid phase was homogenized into theaqueous phase at 75OC containing PoloxamerÆ 188and Tween 80 under a speed rate of 24.000 rpm(UltraTurrax ñ T25, IKA, Labortechnik, Germany)for 3 min. O/W primer emulsion occured at the end

of this stirring time and then this emulsion was ultra-sonicated into bidistilled water at +4OC using anultrasonicator at a power of 30 watts (Bandelin-Sonoplus HD-3100). The obtained emulsion waskept at -20OC for 10 min. and ultrasonicated again at30 watts power to avoid the formation of aggregates.The SLNs were then collected using a Sartorius cen-trifuge filter (Vivaspin 20, molecular mass cut off of10.000 Da) and diluted with 5 mL of water andstored at +4OC for further analysis.

The incorporated amount of candesertan cilex-etil in the supernatant of the SLN dispersion wasdetermined through an indirect method after the cen-trifugation process and the candesertan cilexetil con-centration was measured with a validated UPLCtechnique using a UPLC apparatus (Acquity Waters,UK) equipped with a UV detector set at 304 nm, acolumn oven set at 35 ± 0.5OC, using C8 column(ACQUITY UPLC BEH C8, 1.7 µM, 100 ◊ 2.1 mm,UK) with an injection volume of 10 µL. The mobilephase, fluxed at 0.5 mL/min, was a mixture ofmobile phase A (0.01 M phosphate buffer at pH 3.0)and mobile phase B (100% acetonitrile). The encap-sulated amount of candesertan cilexetil was calcu-lated with the total amount of the active moleculesubtracting free form (5).

The average particle diameter and size distri-bution (PDI value) of the candesertan cilexetilloaded SLN formulations were measured by photoncorrelation spectroscopy (PCS) (Nano ZS, MalvernInst., Malvern, Worcestershire, UK) and the surfacecharge of SLNs was measured by Laser DopplerAnemometry (Malvern Nano ZS, Malvern Inst.,Malvern, Worcestershire, UK). Prior to the meas-urements, the suspended form of nanoparticles wasdiluted in ultrapure water on a dilution of 1/10 (v/v).Five measurements and 25 readings were performedat room temperature for each formulation and themean values were calculated.

The morphology of the candesertan cilexetil-encapsulated SLNs was observed using a FEITecnai G2 Spirit Biotwin Transmission ElectronMicroscopy (TEM; FEI Co., USA). Ten µL of SLNdispersion was placed on a 300 mesh formvar-car-bon coated copper grid (AGAR Scientific, UK) andfixed at room temperature. The sample was stainedwith a 2% uranyl acetate solution for 20 s. The sam-ple was imaged at an accelerated voltage of 80 kV.

In vitro release performance of the developednanoparticles was investigated using a dialysis bagmethod (13). A pH shifting method (2 h in a 0.1 MHCl solution and the remaining period was in pH 6.8phosphate buffer solution to mimic GI tract) anddialysis bags with a molecular mass cut-off of

Development of solid lipid nanocarriers for... 1633

12.000 Da (Sigma-Aldrich) were used as a dissolu-tion medium and dialysis membrane, respectively.Approximately 2 mL candesertan cilexetil-encapsu-lated SLN dispersion was placed into a dialysis bagwith two ends closed by dialysis clamps. Then, thebags were sop firstly in 100 mL of 0.1 M HCl solu-tion for 2 h and after that time mediums werechanged and the bags were sop in 100 mL pH 6.8phosphate buffer solution under sink conditions andcontinously incubated at 37OC in an orbital shakerand vibrated at 100 rpm (Julabo SN 23-2, JULABOGmbH, Seelbach, Germany). Up to 30 days at dif-ferent time intervals, a known amount of releasemedium was collected for UPLC determination and

fresh medium was replaced. The dissolution experi-ments were carried out in triplicate.

A thermal analysis of pure candesertan cilex-etil, CompritolÆ 888 ATO, PoloxamerÆ 188, andthe SLN4 coded SLN formulation was carried outwith a differential scanning calorimeter (DSC)(DSC Q2000, TA Instruments, Likrom, USA).About 2.5 mg of each sample was placed in hermet-ically sealed aluminum pans and analyzed at a scan-ning temperature ranging from 0 to 350OC at a heat-ing rate of 10OC per min.

A statistical analysis was performed usingSPSS Software 20.0 version (SPSS Inc). The resultswere expressed as the mean values ± standard devi-

Figure 1. Encapsulation efficiencies of candesertan cilexetil loaded SLN nanocarriers

Figure 2. Average particle diameters of candesertan cilexetil loaded SLN nanocarriers


ation and statistically analyzed using one-wayANOVA followed by Tukeyís post-hoc test for mul-tiple comparisons. In all cases, an associated p valueof less than 0.05 was considered as statistically sig-nificant.

RESULTS

The encapsulation efficiencies of SLN basednanocarriers are shown in Figure 1. It is seen that theincorporation efficiencies of candesertan cilexetilinto all SLN formulations was found to be very high,in the range of 99.99-100.00 ± 0.011%. This resultobserved that both of the investigated parameters(drug/lipid ratios and surfactant/co-surfactant ratios)

had not significanly affected the entrapment effi-ciencies of the nanoparticles (p > 0.05).

The influence of the drug/lipid ratios and thesurfactant/co-surfactant ratios on the average parti-cle diameter of the SLNs are given in Figure 2. Theaverage particle sizes of all SLN formulationsranged from 90.7 ± 1.46 to 1382.0 ± 83.16 nm.

Figure 3 represents the relation between thePDI values and the surface charge of the candeser-tan cilexetil encapsulated SLN nanocarriers basedon the drug/lipid ratios and the surfactant/co-surfac-tant ratios. In this research, the PDI values of theSLNs indicated a wide size distribution wih a PDIranging from 0.323 ± 0.03 to 0.834 ± 0.06. The zetapotential of the candesertan cilexetil loaded SLN

Figure 3. Effect of drug/lipid and surfactant/co-surfactant ratio on the PDI value and the surface charge of the SLN based nanocarriers

Figure 4. TEM images of SLN1 and SLN4 coded SLN based nanocarriers


formulations was determined to be between 19.5 ±0.6 and 31.5 ± 0.8 mV, with negative surface charge.

TEM images of SLN1 and SLN4 codednanoparticulate formulations, prepared with a con-stant drug/lipid ratio (1/30) and a different surfac-tant/co-surfactant ratio as 1/0.5 and 1/2, respective-ly, are shown in Figure 4. The SLN formulationswere observed in nanometer sizes with sphericalshapes. Aggregation was not seen in TEM images ofthe SLNs.

In vitro dissolution profiles of candesertancilexetil from the SLN formulations are shown inFigure 5. The differences in the release dataobtained from the dissolution studies were statisti-cally compared using a one-way ANOVA test, asimple analysis of variance. The result of this analy-sis indicated significant differences between therelease profiles of drug from the SLNs.

DSC is one of the thermal analysis techniqueswhich is used to designate the physical status of theactive molecule and the excipients and also to con-firm the presence of possible drug-excipientsí inter-actions within the pharmaceutical dosage forms.Figure 6 represents the DSC thermograms of thepure candesertan cilexetil, pure form of CompritolÆ

888 ATO, pure form of PoloxamerÆ 188, and alsocandesertan cilexetil loaded SLN4 coded SLN for-mulation.


Candesertan cilexetil encapsulated SLN for-mulations were prepared using a high shear homog-enization technique followed by an ultrasonicationprocess. The drug/lipid and surfactant/co-surfactantratios were selected as the critical formulationparameters which had been investigated in thisreserach for the design of the efficient colloidalnanocarriers of candesertan cilexetil. In this respect,the developed SLN formulations were evaluated interms of the average particle diameter, PDI values,surface charges, drug-excipient interactions, and invitro release profiles. CompritolÆ 888 ATO wasemployed as a solid lipid matrix material,PoloxamerÆ 188 was selected as the main surfactantand Tween 80 was used as a nonionic co-surfactantin the production process. As seen in Figure 1, thehigh entrapment efficiencies of the active moleculedepended on the highly lipophilic nature of can-desertan. The partition coefficient of the drug at dif-ferent pH values (1.1; 6.9 and 8.9) was detected as >1000 (14, 15). Due to its highly lipophilic structure,candesertan cilexetil was preferred to be locatedhighly into the lipid phase rather than the aqueous

phase during the production process and this situa-tion leads to the complete incorporation of drug intothe solid lipid matrix structure of the nanoparticles.

Figure 2 represented the average particle diam-eters of candesertan cilexetil loaded SLN formula-tions. It was indicated that both of the investigatedformulation parameters significantly affected themean diameter of the solid lipid nanocarriers (p <0.05). At 1/0.5 surfactant/co-surfactant ratio, theaverage particle diameters of the SLN formulationsdecreased when drug/lipid ratio was increased from1/30 to 1/60. This unexpected situation may bereleated to the viscosity differences between thelipid and aqueous phases. Kim et al. (16), reported asimilar result of their study. They explained that thelower average particle diameters were designatedwhen the viscosity differences were higher betweenthe lipid and aqueous phases. When the aqueousphase with lower viscosity was poured into the lipidphase having higher viscosity, bigger lipid dropletswere formed and average particle diametersincreased. On the other hand, when the drug/lipidratio was enhanced from 1/60 to 1/120, the averageparticle diameters of SLN formulations were dra-matically increased due to the predisposition of thelipid to coalescence at high lipid ratios (17). Thisfact can also be derived from the excessivelyincerased viscosity of the lipid phase. Since the highdrug/lipid ratio resulted in increased viscosity of thedispersed phase, bigger lipid droplets occured dur-ing the emulsification process (18). Similar resultswere obtained when the surfactant/co-surfactantratio was enhanced from 1/0.5 to 1/2, for both of theSLN formulations which were prepared with the1/30 and 1/120 drug/lipid ratios (SLN4 and SLN5).It was clearly demonstrated that the average particlediameter of the SLNs significantly decreased withthe increase in the surfactant/co-surfactant ratio (p <0.05). The increase in total amount of the surfactantquantity led to reduction in the interfacial tension ofthe SLN dispersion occured between the lipid andthe aqueous phases during the emulsificationprocess (17, 19).

The relationship between the PDI and the sur-face charge of the SLN formulations of candesertancilexetil based on the drug/lipid ratios and surfac-tant/co-surfactant ratios is presented in Figure 3. AllSLN formulations showed a wide particle size dis-tribution between 0.323 and 0.834. The surfacecharge of the nanoparticles impacted the physicalstability of the nanocarriers. High zeta potential val-ues (≥ 30 mV) indicated high stability of dispersions(20). The zeta potential values of the candesertancilexetil loaded SLN formulations ranged between -


19.5 and -31.5 mV, indicating the stable structure ofdeveloped systems.

TEM images of the candesertan cilexetil incor-porated SLN-based nanocarriers demonstrated thecolloidal size and spherical shapes of the preparedSLN formulations (Fig. 4).

In vitro cumulative dissolution profiles of thecandesertan cilexetil loaded SLNs are given inFigure 5. Significant differences were observedbetween the profiles (p < 0.05). No drug releaseoccurred within 2 h in 0.1 M HCl. On the otherhand, when the medium changed to pH 6.8 phospate

Figure 5. In vitro dissolution profiles of candesertan cilexetil from SLN based nanocarriers

Figure 6. DSC thermograms of the pure and SLN conjugated forms of candesertan cilexetil and all excipients


buffer solution, all of the nanoparticle formulationsexhibited a sustained release pattern of candesertancilexetil after 6th day. The amounts of released can-desertan cilexetil at the end of 30 days were between24.1 and 50.8%, which correlated with the averageparticle diameters of the SLNs. The drug relase fromthe SLN4 coded formulation was significantly fasterthan that of other formulations (p < 0.05). Theseresults demonstrated that when the size of the parti-cles decreased, the drug release rate significantlyincreased from the particles. It is thought that thedecrease in particle size caused a reduction in thelength of diffusion pathways for the active agent.

The drug-excipient interaction is a major prob-lem that occured on the production of the drug deliv-ery systems and the dosage forms. DSC analysis wasperformed to understand the possible interactionsbetween the free drug and the major excipients, suchas the lipids and surfactants, and also the physicalstatus of the incorporated active agent into the carri-er (21). Figure 6 displays the DSC curves of the freecandesertan cilexetil, raw lipid (CompritolÆ 888ATO) and PoloxamerÆ 188, and also the SLN5coded formulation. The DSC curve of the free can-desertan cilexetil shows a sharp exothermic peak at182.78OC, which corresponds to its melting point andindicated the presence of its crystal structure. Theother exothermic peaks which began at 250OC corre-sponded to the oxidative degredation of the activemolecule. These data were previously reported byvarious researchers (5, 6). The melting temperaturesof 79.77OC as a sharp exothermic peak and 52.70OCas a sharp endotermic peak occured for the rawCompritolÆ 888 ATO and PoloxamerÆ 188, respec-tively. The DSC curves of the candesertan cilexetil-encapsulated SLN formulation demonstrated nosharp exothermic peak of the free drug at 182.78OC.This situation indicated that when candesertan cilex-

etil was encapsualted into the lipid matrix structure,the crystal form thereof changed to an amorphousstate. During the production process, the solubility ofthe free drug into the lipid matrix caused a change inthe physical structure of the active agent from crystalto amorphous state (7). During the evaluation of theDSC curves of the SLN formulations, it was seenthat the melting temperatures of the CompritolÆ 888ATO and PoloxamerÆ 188 decreased. Current statemay have originated from the slow evaporation rateor the selective diffusion rate of the organic solventand also the long mixing time of the dispersion dur-ing the production process. Similar results were pre-viously reported by Turk et al. (22).

In conclusion, the combination of high shearhomogenization and ultrasonication methods as aproduction technique has been successfully appliedfor the formation of solid lipid nanoparticles of can-desartan cilexetil. Drug/lipid and surfactant/co-sur-factant ratios strongly affected the final characteris-tics of the SLN-based nanocarriers. Among thedeveloped SLN formulations, SLN4-coded formula-tion containing the lowest drug/CompritolÆ 888ATO ratio and the highest PoloxamerÆ 188/Tween80 ratio was chosen as the best formulation havingimproved dissolution rate of the active drug andhaving the smallest particle size. This research con-cluded that the developed SLN-based nanocarriersmay be considered as an alternative formulation forthe oral delivery of candesartan cilexetil anddeserved further exploration.

Acknowledgments

This research was supported by the ScientificResearch Project Unit of Marmara University(BAPKO; SAG-C-YLP-130213-0035) and ZentivaHealth Products.

Table 1. Compositions of the candesertan cilexetil loaded SLN-based nanocarriers.

CompositionFormulation codes and quantities (g)

SLN1 SLN2 SLN3 SLN4 SLN5

CompritolÆ 888 ATO 1.008 1.008 2.016 1.008 2.016

Candesertan cilexetil 0.034 0.017 0.017 0.034 0.017

Water 42 42 42 42 42

PoloxamerÆ 188 0.504 0.504 0.504 0.504 0.504

Tween 80 0.252 0.252 0.252 1.008 1.008

Drug/Lipid ratio 1/30 1/60 1/120 1/30 1/120

Surfactant/Co-surfactant ratio 1/0.5 1/0.5 1/0.5 1/2 1/2


DECLARATION OF INTEREST

The authors report no conflicts of interest. Theauthors alone are responsible for the content andwriting of this paper.

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Mortality due to cardiac pathologies increaseswith age and hypertension is one of the prevailingissues. Death rate related to hypertension has beenincreased in recent years. Treatment of hypertensionrequires long term therapy and more patient compli-ance. Metoprolol is widely prescribed for the treat-ment of hypertension. It undergoes extensive firstpass metabolism and has short half-life. Frequentdosing reduces patient compliance that can beavoided by using transdermal route (1). For a drug tobe delivered across skin it should have molecularweigh less than 500 Da and log P value from 1 to 3(2). Thus, metoprolol is an ideal candidate for trans-dermal drug delivery system with log P value of 1.6and molecular weight 267.5 Da.

A number of transdermal therapeutic systems(TTS) such as creams, patches, gels etc., are avail-able for systemic delivery of drugs across skin.

However, transdermal patches have an advantageover other systems because of ease of applicationand least cost of manufacturing. Transdermal patch-es are broadly classified as: drug in adhesive (DIA),polymer matrix and reservoir patches (3).

Passive diffusion of drugs across skin is limit-ed due to barrier properties of stratum corneum.Chemical and physical strategies are used toincrease transport through skin by increasing perme-ability of skin and by providing driving force fordrug molecule, respectively (4).

IPM is an aliphatic acid ester. It is a promisingpermeation enhancer for topical formulations and iswidely used as non volatile solvent in cosmetics andpharmaceutical industry.

Aim of the present study was to develophydrophobic transdermal patches of metoprolol byincorporating five different permeation enhancers

PREPARATION, IN VITRO AND IN VIVO CHARACTERIZATION OFHYDROPHOBIC PATCHES OF A HIGHLY WATER SOLUBLE DRUG FOR

PROLONGED PLASMA HALF LIFE: EFFECT OF PERMEATION ENHANCERS

AYESHA YAQOOB, MAHMOOD AHMAD*, ASIF MAHMOOD and RAI MUHAMMAD SARFRAZ

Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Pakistan

Abstract: Aim of present study was to develop metoprolol matrix patches using different enhancers.Combination of two hydrophobic polymers, ethyl cellulose and eudragit RL 100 (8 : 2) were used for prepara-tion of unilaminated matrix patch. 10% w/w of isopropyl myristate (IPM), dimethyl sulfoxide (DMSO), span20 (S20), Tween 20 (T20) and eucalyptus oil as enhancers and 40% of dibutyl phthalate as plasticizer wereused. Prepared patches were evaluated for physical appearance, weight uniformity and thickness. FTIR studieswere performed to assess compatibility among ingredients and developed formulation. Dissolution and perme-ation studies were performed to compare effects of enhancers. Surface morphology after release was examinedby scanning electron microscopy. Selected formulation was subjected to in vivo studies by randomizedcrossover design in rabbits (n = 6) for pharmacokinetic comparison with oral solution administration. Physicalevaluation revealed that translucent, flexible, non brittle patches of uniform weight and thickness were pre-pared. Release from patches followed Higuchi model. Mechanism of release was Fickian. Formulation con-taining IPM showed that release was by anomalous transport. Highest permeation flux was observed for for-mulation containing IPM with 2-fold enhancement in permeation. Permeation flux for patches was in order offormulation with no enhancer > IPM > T20 > S20 > DMSO = eucalyptus oil. Plasma concentration from in vivostudies exhibited sustained plasma levels of metoprolol after transdermal patch application in comparison tooral solution administration. Pharmacokinetic analysis of in vivo data elucidated that half life was increased 8times when compared to oral administration, due to controlled release of drug for longer period of time. Thesefindings suggested that hydrophobic transdermal patches of highly water soluble drug metoprolol were suc-cessfully prepared with 10% of IPM for sustained systemic delivery for prolonged half life.

Keywords: isopropyl myristate, metoprolol, transdermal patch, hydrophobic, permeation enhancers, skin dif-fusion, in vivo

1639

* Corresponding author: e-mail: [email protected]; mobile: 0092300-9682258; fax: 0092-062-9255565

1640 AYESHA YAQOOB et al.

and two hydrophobic polymers i.e., ethyl celluloseand eudragit RL 100 for transdermal delivery ofmetoprolol.

EXPERIMENTAL

Materials

Metoprolol tartrate was received as a generousgift from Acto Laboratories, Karachi, Pakistan.Eudragit RL 100 (M/s Rohm Pharma GmbH,Darmstadt, Germany), ethyl cellulose (ethoxy con-tents 47.5-49% w/w; viscosity 10 cps, SigmaAldrich, USA), ammonium dihydrogen phosphate(AppliChem, Germany), dichloromethane (FisherScientific, USA), polyvinyl alcohol (m.w. 75 000,AppliChem, Germany) were purchased. Isopropylmyristate, eucalyptus oil, dimethyl sulfoxide werepurchased from Aldrich Sigma (USA). Ortho-phosphoric acid, dibutyl phthalate, chloroform, span20, Tween 20, micro filters of 0.45 micron, andHPLC grade acetonitrile and methanol were pur-chased from Merck (Germany). All other chemicalsused were of analytical grade.

Preparation of patches

Patches were prepared by plate casting method.At first, PVA backing membrane was fabricated bypreparing 4% w/v clear solution of PVA at 90OC onhot plate magnetic stirrer. Five mL of PVA solutionwas poured in glass Petri dishes of 18.5 cm2 areawith the help of graduated pipette and evaporated inoven at 40OC for 24 h. After drying, backing mem-brane was not removed from Petri dishes. In secondstep, 250 mg of ethyl cellulose (EC) and eudragit RL100 (ERL) in ratio of 8 : 2 were dissolved in 10 mLof chloroform by sonication. Eighty mg of metopro-lol, 40% w/w of dibutyl phthalate (100 mg) and 10%w/w of enhancer (25 mg) were added in clear poly-meric solution. This clear matrix solution waspoured on Petri dishes containing transparent back-ing membrane and allowed to evaporate underinverted funnel for 12 h at 40OC. Dried films weretaken out from Petri dishes and cut in circular piecesof 1.5 mm diameter for further analysis. Patcheswere evaluated for physical appearance, weight andthickness variation.

Fourier transform infrared (FTIR) spectroscopy

FTIR studies were conducted to assess com-patibility between drug and excipients. FTIR spectraof drug, polymers and prepared transdermal filmswere recorded using attenuated total reflectance(ATR) Bruker FTIR (Tensor 27 series, Germany)apparatus with scanning range of 4000-600 cm-1.

Raw material was grounded for uniformity of parti-cle size before analysis and already prepared thintransparent films were directly scanned and ana-lyzed by using Opus data collection software toidentify any physical and chemical interaction.

Dissolution studies

Dissolution of patches was performed onPharmatest apparatus attached with automated frac-tion collector (Pharma Test, Germany) by using pad-dle over disk method (U.S.P. apparatus no. 5). Discassembly was prepared with stainless steel mesh ofpore size 125 µm, paper clips and watch glass.Phosphate buffer pH 7.4 was used as dissolutionmedium (5). Disk assembly with releasing surfacefacing upward was placed in glass dissolution vesselcontaining 500 mL of phosphate buffer. Temperaturewas maintained at 32 ± 1OC and media were stirredwith paddles at speed of 50 rpm (6). Disk assemblywas designed to reduce dead volume between ves-selís bottom and patch holder and to kept patchrelease surface parallel to paddle blade bottom.Distance between disc and paddle blade was kept atalmost 25 mm. Samples were withdrawn at timepoints 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24 h.

Skin diffusion studies

For diffusion studies, prepared rabbit skin wasmounted between two compartments of Franz diffu-sion cell (PermeGear, USA). Receptor compartmentwas filled with phosphate buffer solution pH 7.4 andkept on stirring on multi plate magnetic stirrer (7).Temperature in receptor compartment was main-tained at 32 ± 1OC by automated thermostat. Patch ofan area 1.77 cm2 was placed on skin in donor com-partment. Samples were taken from sampling port atpredetermined time intervals.

Absorbance of each sample was determinedwithout any further dilution on UV-Vis spectropho-tometer (IRMECO U2020, Germany) at λmax of 223nm. Samples of skin penetration study were ana-lyzed after appropriate dilution with phosphatebuffer. Phosphate buffer pH 7.4 was used as blank.Concentration of drug at each sampling point wascalculated from calibration curve.

Elucidation of drug release mechanism

Dissolution and skin diffusion data were sub-jected to various kinetic models by using DDSolverÆ Excel based ads in program (8) to elucidaterelease mechanism from patches. Zero order kinet-ics indicates that drug release rate is independent ofthe amount of drug in formulation.

Ft = Kot

Preparation, in vitro and in vivo characterization of hydrophobic patches of... 1641

First order kinetic model indicates dependencyof drug release on amount of drug in formulation attime t.

K1tlog Ft = log Fo + ñññññññññ2.303

Higuchi plot deals with release of drug fromuniform matrix formulation by diffusion. Thismodel is used to describe release of water solubledrug from transdermal patches and modifiedreleased formulations (9).

Ft = KHt1/2

Korsmeyer Peppas kinetic model is expressedby following equation:

Ftñññ = Kptn

Fo

where Ft is amount of drug released at time t and Fo

indicates the amount of drug added in formulation.Ft/Fo is the fraction of metoprolol released. Ko, K1,KH and Kp are release constants of zero order kinet-ics, first order model, Higuchi plot and Korsmeyermodel, respectively. ìnî is release exponent ofKorsmeyer Peppas plot and elucidate the behaviorof diffusion release. As value of ìnî approaches 0.5,the release mechanism follows Fickian diffusion.When 0.5 < n > 1, the mechanism of diffusionrelease is anomalous. If n = 1, the release is Case-IItransport (10).

Patch surface morphology studies

Patch surface was evaluated visually forsmoothness and homogeneity before release study.The behavior of transdermal films after metoprololrelease was observed under scanning electronmicroscope (SEM). Small pieces of patch afterrelease were directly scanned under electron micro-scope (Quanta 250) using maker FEIÆ software.Surface morphology of patches further explaineddrug release mechanism.

In vivo studies in rabbits

Patch having the highest permeation flux wasselected for in vivo studies in rabbits. It was acrossover single dose study. Six rabbits of weight1.5-2 kg were obtained from pharmacology animalhouse. Weight of each rabbit was noted and animalswere properly labeled for identification. One daybefore experiment rabbits were kept fastingovernight but had free access to water. Twenty mgof metoprolol in form of solution was administeredto rabbits with a plastic syringe (11). Blood sampleswere taken in EDTA tubes at time points of 0, 0.5,1, 2, 3, 4 and 6 h. Plasma was separated by centrifu-gation at speed of 5000 rpm. Transdermal applica-tion of metoprolol patch was done after one week of

washout period. One day before transdermal patchapplication, hairs were removed from abdominalregion of rabbits with depilatory cream (AnneFrench, Pakistan). After hair removal, skin wascarefully washed with distilled water. On the day ofexperiment, patch containing 80 mg of metoprololwas applied on hair free area with adhesive tape.Blood samples after transdermal application weretaken at 0, 1, 3, 6, 9, 12, 24, 36 and 48 h. Plasma wasseparated and stored at -70OC. On the day of analy-sis, plasma was allowed to come at room tempera-ture. Plasma samples were prepared by liquid-liquidextraction with dichloromethane (1ñ2 mL) and con-centrated on sample concentrator at 40OC. Two hun-dred µL of mobile phase was used to reconstituteplasma samples. Two hundred µL of these preparedsamples were analyzed by HPLC on AgilentTechnologies series 1100 (USA) chromatographwith a pump and UV-Vis detector by using BDSHypersil C18 column (250 ◊ 4.6 mm, particle size 5.0µm, Thermo Electron Co., USA). Mobile phase wasa combination of ammonium dihydrogen phosphate(50 mM) solution and methanol (50 : 50, v/v) adjust-ed to pH 3.05. Concentration of drug in plasma wasdetermined by using calibration curve plotted byconcentration versus peak area of drug in chro-matograms of spiked plasma. In vivo data were sub-jected to pharmacokinetic analysis by using applica-tion package KineticaÆ version 5.0 (Thermo FisherScientific, USA).


Physical characterization

Physical characterization of preparedhydrophobic patches of metoprolol was done visual-ly. All patches showed reasonable tensile strengthand were non-brittle. Surface was found smooth inappearance. Patches without enhancer as well ascontaining enhancers were translucent in their phys-ical appearance. Smoothness, physical strength andtransparency were increased due to the presence ofenhancers. Backing membrane was firmly attachedwith matrix layer. However, improper storage formore than six months resulted in detachment ofbacking membrane and brittle patches. Weight/cm2

and thickness were found uniform throughout pre-pared polymeric films.

FTIR spectra analysis

FTIR spectra of pure drug, polymers and pre-pared patches containing enhancers were recordedto assess physical and chemical interactions betweendrug and excipients. The most characteristic peaks


of metoprolol spectra were at 820.53, 1108.28 and1512.50 cm-1. All these characteristic peaks of puredrug were also present in the spectra of all preparedhydrophobic films containing drug (with or withoutenhancer). Selected FTIR spectra are shown inFigure 1. Thus, the FTIR spectra analysis indicatedthat no interaction occurred between metoprolol andfilm ingredients during formulation. The most char-acteristic peak of prepared films was at 1725 cm-1

which was observed in all formulations with orwithout enhancers.

Dissolution studies

Dissolution studies were performed to evaluateeffects of enhancers (10% w/w) on release of drug.

Amount of metoprolol released was plotted againsttime and released profile of patches with enhancerand without enhancer is presented in Figure 2. Initialburst release was observed for all formulations. Afterinitial fast release of surface drug, release becameslower. The initial burst release will be helpful toachieve therapeutic plasma levels in short time (12).Incorporation of enhancers had not affected amountof drug release up to 24 h from patches except in for-mulations containing DMSO. DMSO containing for-mulation showed more than 90% drug release within12 h. All other formulations showed less than 90% ofdrug release (85.62 to 88.91%) up to 24 h.

Release pattern was not altered for the formu-lations containing enhancers. However, presence of

Figure 1. FTIR spectra of metoprolol (MET), EC, ERL and patch containing IPM (P-IPM)


enhancers affected the burst release properties ofpatches. The highest burst effect was observed forformulation containing DMSO with T50% (timetaken to release 50% of drug) of 2.18 h as shown inTable 1.

T50% is an indicator of fast release of drug (13).Less initial burst release was observed for formula-tion containing span 20 with T50% of 8.14 h. Thelowest value of T90% (time to release 90% of drug)was observed for formulation containing DMSO.No significant statistical difference (p > 0.05) wasobserved between values of T90% of other formula-tions.

DMSO is commonly used as co-solvent in top-ical formulations. It is also called as ìuniversal sol-ventî. It has polar nature and has ability to cross bio-logical membranes. Due to solubilizing ability it is

used as enhancer for topical and transdermal drugdelivery. Smaller values of T50% and T90% can berelated to co-solvent property of DMSO.

To elucidate mechanism of release, data weresubjected to release kinetic models. Release param-eters are shown in Table 1. R value (correlationcoefficient) was found in order of KorsmeyerPeppas > Higuchi > first order > zero order. Asrelease followed Korsmeyer Peppas and Higuchimodel, thus release was by diffusion of highly watersoluble drug from matrix of hydrophobic patch.Value of release exponent ëní helps to understandbehavior of diffusion release. Formulations, exceptthat containing IPM, had ëní less than 0.5, thusrelease was by Fickian diffusion. For formulationcontaining IPM the value of ëní was 0.528, sorelease followed anomalous transport i.e., non-

Figure 2. Metoprolol release from patches containing 10% of various enhancers. S 20 = span 20, T 20 = Tween 20

Table 1. Release kinetics of metoprolol from patches.

Enhancer T50% (h) T90% (h)Zero order First order Higuchi's plot Korsmeyer Peppas

R R R KH R n

No enhancer 6.333 27.29 0.9318 0.9880 0.9927 18.764 0.9970 0.414

Eucalyptus oil 5.142 26.49 0.9310 0.9870 0.9918 20.124 0.9970 0.394

IPM 7.435 28.74 0.9475 0.9969 0.9899 18.612 0.9896 0.528

S 20 8.138 25.34 0.9700 0.9779 0.9920 17.286 0.9913 0.467

T 20 5.800 25.19 0.9371 0.9786 0.9552 19.220 0.9929 0.396

DMSO 2.188 12.43 0.8395 0.9926 0.9499 24.402 0.9813 0.308

S 20 = span 20, T 20 = Tween 20


Fickian mechanism of diffusion. High values ofHiguchi rate constant (KH) was observed for patchcontaining DMSO. Thus, release from patches wassustained and diffusion controlled.

Patch surface morphology studies

Surface morphology of patches after drugrelease was examined under electron microscope.SEM images of patch containing no enhancer andthat of containing IPM as enhancer are shown inFigure 3. The image of patch containing no enhancershowed unequal distribution of pores of differentsizes. Patch containing IPM showed only largepores indicating that erosion mechanism of releasewas also involved in drug release. These resultswere also supported by release exponent ìnî of IPMcontaining patch, indicating non-Fickian mechanismof diffusion. Smaller pores were less in patch con-taining IPM.

Skin diffusion studies

Skin diffusion studies help to predict in vivoperformance of transdermal patch. Aim of skin dif-fusion studies was to evaluate effects of 10% per-meation enhancers on diffusion of metoprololthrough excised rabbit skin. Amount of drug perme-ated (µg/cm2) was plotted against time (h) as shownin Figure 4. Permeation parameters i.e., cumulativeamount of drug permeated up to 30 h (Q30, µg/cm2),permeation flux at steady state (Jss, µg/cm2/h) andenhancement ratio (ER) are given in Table 2. Bar

Figure 4. Metoprolol permeation through rabbit skin from patches containing 10% of various enhancers. S 20 = span 20, T 20 = Tween 20

Figure 3. SEM photographs of (A) patch without enhancer and(B) patch containing 10% IPM


chart of permeation flux of formulations containingdifferent enhancers is shown in Figure 5.

All enhancers containing formulations showedsignificantly higher (p < 0.05) values of permeationflux through rabbit skin as compared to formulationswithout enhancer. Highest permeation flux of 65.03

± 33.50 µg/cm2/h was observed in formulation con-taining IPM with 2.12 folds enhancement in perme-ation as compared to control (no enhancer). IPM hasalso exhibited highest value of Q30 (2277.99 ±958.21). Formulation containing Tween 20 showed2.05 folds rise in permeation flux. Enhancement of

Figure 5. Bar chart of permeation flux of metoprolol through rabbit skin from patches containing various enhancers

Figure 6. Plasma concentration-time profile of metoprolol after transdermal and oral administrations in rabbits (n = 6)


permeation induced by various enhancers was indecreasing order of IPM > Tween 20 > Span 20 >DMSO = eucalyptus oil (p = 0.292).

Results obtained in present study are supportedby the findings of Yousuf et al., whereas IPM wasevaluated as enhancer for combination of drugs:ketotifen fumarate and salbutamol sulfate (14). IPMhas been declared best permeation enhancer inanother work conducted by Das et al., where it wascompared with another aliphatic ester isopropylpalmitate (IPP) for permeation enhancement of tra-zodone in a matrix patch (15).

IPM is well tolerated permeation enhancer fortopical formulations and is widely used as nonvolatile solvent in cosmetic and pharmaceuticalindustry (16). IPM disrupts highly order lipid struc-ture of skin thus altering metoprolol permeabilityacross skin. IPM is lipophilic in nature thus has innateability to interact with lipid bilayers. IPM can alsopartition into polar phase (proteins) of skin because ofits intermediate polar nature (15, 17). IPM belongs toclass of aliphatic esters and according to Sato et al.this class of penetration enhancers act both by

increasing partition coefficient of metoprolol for skinas well as by increasing its diffusivity to skin (18).

In present study, Tween 20 was found to bemore effective when compared to span 20. Span 20and its ethoxylate derivative polysorbate 20 (Tween20) belongs to class of non ionic surfactants. Bothhave same C12 fatty acid chain but differ in theirpolar head functional groups. Tween 20 is morehydrophilic than span 20. Being hydrophilic bothnon ionic surfactants exert their effects by anincrease in skin hydration, resulting in increase indrug partitioning across skin and leading to higherpenetration of metoprolol through rabbit skin.

Results of permeation studies showed thatDMSO and eucalyptus oil were equally effective formetoprolol with respect to penetration enhancement.Eucalyptus oil was found as effective permeationenhancer (30-fold increase in permeation) for anoth-er highly water soluble drug 5-florouracil in a studyby William and Barry (19). Effects of DMSO aspenetration enhancer can be related to its lipidextraction efficiency as well as partitioning increas-ing property (20).

Table 2. Permeation parameters and kinetics of metoprolol through rabbit skin from patches.

Enhancer Q30 Jss ER Zero order First order Higuchi model

No enhancer 1309.03 ± 379.47 30.60 ± 10.98 - 0.9595 0.9677 0.9926

Eucalyptus oil 2040.94 ± 780.61 54.42 ± 24.67 1.78 0.9804 0.9818 0.9821

IPM 2277.99 ± 958.21 65.03 ± 33.50 2.12 0.9886 0.9881 0.9794

S 20 2055.80 ± 900.60 55.32 ± 24.59 1.81 0.9817 0.9852 0.9860

T 20 2157.23 ± 754.59 62.67 ± 26.45 2.05 0.9837 0.9783 0.9631

DMSO 2143.67 ± 1051.04 51.90 ± 24.30 1.70 0.9703 0.9828 0.9973

S 20 = span 20, T 20 = Tween 20

Table 3. Pharmacokinetic parameters (mean ± S.D.) of metoprolol in rabbits (n = 6) plasma after oral solutionadministration and transdermal patch application.

Pharmacokinetic Oral Transdermal parameters solution patch

Cmax (ng/mL) 437.94 ± 137.53 91.61 ± 11.61

T max (h) 0.50 ± 0.00 9.00 ± 1.77

AUC 0-t (ng h / mL) 822.96 ± 213.12 2327.95 ± 49.03

AUC0-∞ (ng h / mL) 1140.06 ± 269.90 4387.73 ± 831.65

Css 137.16 ± 35.52 48.50 ± 1.02

MRT (h) 4.71 ± 0.60 46.58 ± 6.92

t 1/2 (h) 3.71 ± 0.56 30.22 ± 5.85

% Relative bioavailability 96.22


Results of kinetic analysis of permeation dataare given in Table 2. Best fit equation for kineticanalysis of permeation data was in order of Higuchi> first order > zero order for formulation containingeucalyptus oil, DMSO, span 20 and for that contain-ing no enhancer. Formulations containing Tween 20and IPM, values of R2 were in decreasing order ofzero order > first order > Higuchi. These resultswere due to skin barrier which has slowed the per-meation of drug leading to almost zero order perme-ation.

In vivo studies

Formulation containing 10% of IPM wasselected for in vivo studies in rabbits (n = 6). Plasmaconcentration-time profile after metoprolol oralsolution administration and transdermal patch appli-cation is shown in Figure 6. Metoprolol wasdeclined faster after oral administration, whereasplasma concentration remained relatively stable forlonger period of time after transdermal application.Pharmacokinetic parameters of both treatments aregiven in Table 3. Statistical analysis of in vivo datawas performed and pharmacokinetic parameterswere found significantly different (p < 0.05) afteroral and transdermal administration. Maximumplasma concentration (Cmax) after oral administrationwas 437.94 ± 137.53 ng/mL, in comparison to 91.61± 11.61 ng/mL for transdermal application. Time toreach maximum plasma concentration (Tmax) was 0.5h after oral administration whereas after transdermalapplication it was 9 h. This lower value of Cmax andhigher value of Tmax after transdermal application of80 mg dose showed that drug was released at sus-tained level from patch for longer period of time.Mean residence time (MRT) after transdermaladministration (46.58 ± 6.92 h) was high in compar-ison to oral administration (4.71 ± 0.60 h). Plasmahalf life (t1/2), an indicator of prolonged drug deliv-ery, was extended from 3.71 ± 0.56 h after oraladministration to 30.22 ± 5.85 h after transdermalapplication. Relative bioavailability after transder-mal patch application was 96.22%.

Oral administration of 20 mg of metoprololresulted in higher value of Cmax whereas Cmax in caseof transdermal application of 80 mg patch waslower. However, steady state plasma concentration(Css) with no plasma fluctuation resulted in transder-mal administration for prolonged period of time asindicated from t1/2 and MRT and this will be helpfulin avoiding side effects related to frequent oraladministration. The higher value of MRT was due tocontinuous delivery of drug from patch present onskin in to systemic circulation.

Rama et al. (21) performed comparative phar-macokinetic study of another β blocker - propranololhydrochloride in rabbits (weight = 1.2-1.8 kg) afterapplication of patches of an area 18 cm2 containing30 and 45 mg drug (for 30 h). Oral solution wasadministered in dose of 10 mg/kg (6 h). In theirstudy MRT was found 5 times higher than that oforal administration, whereas in the present study,MRT after transdermal patch application was found18 times higher than that of oral administration (21).

In vitro release and permeation studies revealedslow sustained delivery of drug from hydrophobicpatches. When tested in rabbits, this slow and sus-tained drug delivery resulted in continuous and pro-longed absorption of metoprolol through skin in tothe systemic circulation.

CONCLUSION

Present study concludes that combination oftwo hydrophobic polymers EC and ERL can providediffusion controlled release of metoprolol and high-est permeation flux across excised rabbit skin in thepresence of 10% w/w of different enhancers.Controlled and sustained drug delivery resulted incontinuous and prolonged systemic absorption ofhighly water soluble drug metoprolol for prolongedplasma half life.

Acknowledgment

Authors are really thankful to higher educationcommission (HEC) of Pakistan for providing finan-cial support for this study in form of indigenousscholarship.

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Received: 27. 04. 2016


Currently, there is growing number of studieson the treatment and relapse prevention of tumorfocused on the inhibition of cyclooxygenase-2(COX-2) activity and decrease the prostaglandin E2

(PGE2) level in tumor cells (1-3).Flurbiprofen axetil is a non-steroidal anti-

inflammatory drug that inhibits COX-1 and COX-2non-selectively. It is widely used in preemptiveanalgesia, postoperative pain and cancer pain thera-py (4).

At present, there is no reported study that indi-cates whether flurbiprofen axetil can inhibit thegrowth of tumor tissue of cervical cancer, andwhether PGE2 and COX-2 are involved in tumorgrowth mechanism of cervical cancer.

Thus, the aim of this study was designed toinvestigate whether flurbiprofen axetil inhibits thegrowth of tumor tissue of cervical carcinoma andaffects the content of PGE2 in tumor tissue.


Experimental animals and groups

Fifty female BALB/c nude mice were selected.Their age- and weight-range was 4-6 years and 18-22 g. The animals were fed in specific pathogen free(SPF) animal lab, and kept a constant humidity andtemperature. The food, cage and bedding were ster-ilized. After one week of adapting the environment,the nude mice were used for tumorigenicity experi-ment. The animals were randomly divided into con-trol group (C), tumor + saline group (T), tumor +flurbiprofen axetil 10 mg/kg (Cf10) group, tumor +flurbiprofen axetil 25 mg/kg (Cf25) group, tumor +flurbiprofen axetil tumor 50 mg/kg (Cf50). Tenmice were kept in each group. In the control group,the peritoneal cavity was injected with saline forconsecutive 15 days; while in the experimentalgroup, before 1 day of modelling, the peritoneal cav-

PHARMACOLOGY

THE INVESTIGATION OF EFFECT OF FLURBIPROFEN AXETIL ON THE TISSUE GROWTH AND THE CONTENT OF PGE2 IN CERVICAL CANCER

JING LU1,2, SHENGGANG WANG2, GUIYING CHEN2, XIAOFENG SUN2 and KEZHONG LI1,*

1Department of Anesthesiology, The Second Hospital of Shandong University, Jinan 250033, Shandong Province, P. R. China

2Department of Anesthesiology, Linyi Peopleís Hospital, Linyi 276003, Shandong Province, P. R. China

Abstract: The aim of this study was to investigate whether flurbiprofen axetil can inhibit the tissue growth andthe content of PGE2 in cervical cancer or not. Fifty female BALB/c nude mice were randomly divided into con-trol group (C), tumor + saline group (T), tumor + flurbiprofen axetil 10 mg/kg (Cf10) group, tumor + flur-biprofen axetil 25 mg/kg (Cf25) group, tumor + flurbiprofen axetil tumor 50 mg/kg (Cf50), so that each grouphad 10 animals. Then, the animal model of human cervical carcinoma was established, and the relative tumorvolume (RTV), relative tumor proliferation rate (T/C) and tumor inhibition rate were measured. The content ofPGE2 in tumor tissue was determined by using enzyme-linked immunosorbent assay. There was no tumor for-mation in group C, and the time of tumor growth in other groups was non-statistically different. The RVT inCf50 group was lower than in other groups. It was evident from the curve of tumor growth that the tumor weightin T group was evidently higher than that of administration groups (p < 0.01). The tumor inhibition rates ofCf10, Cf25 and Cf50 groups were 16.8, 19.6 and 36%, respectively, and the relative tumor proliferation ratewere 85, 91 and 72%, respectively. The PGE2 level of Cf50 was statistically (p < 0.01) lower than that of Cf10and Cf25 groups. Flurbiprofen axetil can inhibit the growth of cervical cancer transplanted tumor in nude miceand this inhibitory effect was maximal in Cf50 group. Flurbiprofen axetil can inhibit the production of PGE2 intumor tissue of cervical carcinoma in nude mice.

Keywords: flurbiprofen axetil, tumor, PGE2

1649


1650 JING LU et al.

ity was injected with flurbiprofen axetil and salinefor consecutive 15 days. Control group and experi-mental groups received different dosage flurbipro-fen axetil injection twice a day (at 8:00 a.m. and8:00 p.m.). On the 15th day, all animals were killedin the experimental groups. During the experiment,the disposal of animals was in accordance with theinstructive notions with respect to caring for labora-tory animals promulgated by Ministry of Scienceand Technology in 2006.

Establishment of the animal model of cervical

cancer

The experimental animals and tumor cell linesSPF female (BALB/C) nude mice (4~5 years old)with the weight of 16~20 g were purchased fromAier Matt Technology Co., Ltd. in Suzhou IndustrialPark under Suzhou University Laboratory AnimalsCenter [license number: SYXK (Su) 2007-0035]and fed in the Animal Research Center, as well asachieved the condition of SPF during the process ofexperiment.

Then, the Hela cells at the logarithmic stage ofgrowth were taken, and adjusted the cell density at 1◊ 107/mL. Then, 100 µL cell suspension was inject-ed to left axilliary vein of nude mice, and observedthe situation of the tumor growth. The diameter ofthe subcutaneous nodules > 0.5 cm was taken as thestandard of tumor formation.

Detection index

Calculation of volume measurement of tumors andtumor inhibition rate

On the administration day and every 3 daysafter administration, the length- (a) and width-diam-eter (b) of tumor were measured by using Verniercaliper. At the end of administration, stripping of thetumor for weight (WT) was conducted. The follow-ing formulas were used to calculate the tumor vol-ume, relative tumor volume, relative tumor prolifer-ation rate and tumor inhibition rate:

Tumor volume = ab2/2Relative tumor volume (RTV) = Vt/V0

where, Vt is the tumor volume at the end of experi-ment and V0 is the tumor initial volume.Relative tumor proliferation rate (T/C, %) = RTV

treated group/ RTV control group ◊ 100%Tumor inhibition rate = [(Average tumor weight ofcontrol group - Average tumor weight of adminis-tration group)/ Average tumor weight of control

group] ◊ 100%

The observation time (d) was taken on the hor-izontal axis, mean tumor volume (mm3) as the verti-cal axis for the drawing of tumor growth curve.

Measurement of PGE2 content in the tumor tissueby using enzyme-linked immunosorbent assay

The tumor tissue of nude mice was homoge-nized and the supernatant was taken to test using kitto measure the PGE2 content in the tumor tissue byusing enzyme-linked immunosorbent assay.

Statistical methods

The SPSS version 19.0 was used. All data wereexpressed as the mean ± standard deviation (x ±SD). The data from various groups were comparedby using q-test (Newman-Keuls) and LSD method.The c2 test was used for the comparison of data andrate; p < 0.05 was used for statistically significantdifferences.

RESULTS

Time of tumor formation

No tumorigenicity was noted in group C. Thetime of tumor formation in T group, Cf10 group,Cf25 group and Cf50 group was 7.6 ± 0.8 days, 7.0± 0.9 days, 7.3 ± 0.9 days and 6.7 ± 0.9 days, respec-tively. There was non-statistically significant (p >0.05) difference between the groups.

Table 1. Growth state of cervical cancer tumor and contents of PGE2 in tumor tissue was compared among groups (x ± s).

Time of tumor Tumor Contents of Groups formation

VT RTV

WTinhibition PGE2

(days)(mm3) (g)

rate (%) (ng/g)

C 7.6 ± 0.8 928.8 ± 54.5 11.0 ± 1.9 1.01 ± 0.11 / 1.94 ± 0.21

Cf10 7.0 ± 0.9 773.3 ± 83.0 * 9.3 ± 1.2 0.84 ± 0.06* 16.8 1.52 ± 0.11*

Cf25 7.3 ± 0.9 769.1 ± 55.0 * 9.9 ± 0.8 0.81 ± 0.07* 19.6 1.46 ± 0.18*

Cf50 6.7 ± 0.9 596.6 ± 83.8 *∆ 7.8 ± 2.0 * 0.65 ± 0.05*∆ 36 ∆ 0.9 ± 0.16*∆

* p < 0.01 compared with C group; ∆ p < 0.01 compared with Cf25 group.

The investigation of effect of flurbiprofen axetil on the tissue... 1651

Relative tumor volume

There was non-statistically significant (p >0.05) difference among the RTV values of experi-mental groups. The RVT values of each group areshown in Table 1. The results indicated that the RVTvalues of T group were all significantly (p < 0.01)higher than that of Cf groups; while the RVT valuesof Cf50 group were all significantly (p < 0.01) lowerthan that of Cf25 group (p < 0.01). In addition, therewas non-statistically significant (p > 0.05) differ-ence between the values of Cf10 and Cf25 group.

Tumor inhibition rate

The curve of tumor growth is shown in Figure1. The results of tumor weight at the end of experi-ment are shown in Table 1, which shows that thetumor weight of T group was significantly (p < 0.01)higher than that of administration group. The tumorweight of Cf50 group was significantly (p < 0.01)higher than that of Cf10 and Cf25 group. There wasnon-statistically (p > 0.05) significant differencebetween Cf10 and Cf25 group.

Content of PGE2 in tumor tissue

The contents of PGE2 in tumor tissue of eachgroup are given in Table 1. The results indicated thatthe PGE2 level of T group was significantly (p <0.01) higher than that of the administration group.The PGE2 level of Cf50 group was significantly (p <0.01) higher than that of Cf10 and Cf25 group.There was non-statistically (p > 0.05) significantdifference between the values of PGE2 for Cf10 andCf25 group.

DISCUSSION

Effect of cyclooxygenase on the growth and

development of tumor

Cyclooxygenase (COX) is a rate-limitingenzyme in the synthesis of prostaglandins (PGS)during the process of arachidonic acid metabolism,including COX-1 and COX-2 two isoenzymes. Inrecent years, the research has indicated that theCOX-2 is not only the central part of the inflamma-tory response, but also has a close relationship withthe occurrence and development of tumor (1).COX-2 is an inducible cyclooxygenase, which israpidly activated by different kinds of damages ofchemical, physical, and biological nature. It cat-alyzes the synthesis of prostaglandins, and isinvolved in the inflammatory response. There isalmost no expression of COX-2 in normal tissues;however, COX-2 is overexpressed in the conditionsof stress or cytokines incentives. The activity ofCOX-2 is enhanced with an increase in PGE2 leveland thereby participates in the process of tumor for-mation (2, 3).

Flurbiprofen axetil inhibits the growth of cervical

cancer xenograft

Cyclooxygenase-2 (COX-2) inhibitors arenewly investigated drugs for the treatment and pre-vention of cancer. However, the cardiovascular sys-tem is affected with the event of COX-2 inhibition,i.e., COX-2 inhibition may result in hypertension,coronary heart disease and myocardial infarction insome patients. Flurbiprofen axetil is an NSAID,

Figure 1. Curve of tumor growth

1652 JING LU et al.

which has been widely used in preemptive analge-sia, postoperative pain and cancer pain therapy. Theresults of this study showed that flurbiprofen axetilcan inhibit the growth of cervical cancer xenograftsin nude mice and Cf50 is the best in inhibitoryeffect. Moreover, there was no adverse reaction inany group. Flurbiprofen axetilís anticancer mecha-nism may be associated with the following aspects:(i) because of its targeting feature, i.e., it has a littleeffect on normal cells since it selectively inhibits thetumor cells; (ii) by inhibiting the activity of COX-2,the generation of PGE2, TXA2, and PGS is reducedand the vascular endothelial growth factor (VEGF)and basic fibroblast growth factor (bFGF) release isblocked leading to the inhibition of tumor angiogen-esis (7); (iii) it enhances the response to radio-therapy and chemotherapy of tumor cells. Thestudy from Sorensen has indicated that the lowdose of celecoxib can evidently enhance the cyto-toxicity of bleomycin, cisplatin and vincristine;and (iv) it strengthen the effect of tumorimmunotherapy (5, 8-10).

Mechanism of PGE2 participate in the inhibition

of cervical cancer xenografts by flurbiprofen

axetil

PGS have extensive and important biologicalactivity, which is not only involved in the inflam-matory response and in cancer occurrence, but alsoplays an important role in the generation and devel-opment of tumor, and PGE2 is most active in thisregard. The studies have shown that over-expressionof COX-2 is one of the reasons to promote tumori-genesis. Moreover, the over-expression of COX-2can promote the synthesis of PGE2 (11). With anexcessive number of PGE2 in tumor cells, the prolif-eration and invasion of tumor cell can be promoted(12). The results of this study showed that (i) PGE2

level in tumor tissue of cervical carcinoma in nudemice significantly increased; and (ii) flurbiprofenaxetil can inhibit the growth of cervical cancerxenografts in nude mice and the content of PGE2 intumor tissues are significantly decreased. Under theexperimental conditions of this study, the mecha-nism of flurbiprofen axetil to inhibit the growth ofcervical cancer xenografts in nude mice could be

related to the generation of PGE2. Thus, specificinhibitors of PGE2 may be a new type of targets forcancer treatment and prevention.

CONCLUSION

Flurbiprofen axetil can inhibit the growth ofcervical cancer transplanted tumor in nude mice andthis inhibitory effect was maximal in Cf50 group.Flurbiprofen axetil can inhibit the production ofPGE2 in tumor tissue of cervical carcinoma in nudemice.

Acknowledgment

This research is supported by Health Medicineand Science Technology development plan, ShandongProvince, China. Project number: 2013WS0076.

REFERENCES

1. Kamei D., Murakami M., Nakatani Y.: J. Biol.Chem. 278, 19396 (2003).

2. Julie G., Jaffer A.: J. Clin. Oncol. 25, 4865(2007).

3. Grosch S., Maier T.J., Schiffmann S.: Natl.Cancer Inst. 98, 736 (2006).

4. Shen J., Gan L., Zhu C.: Int. J. Pharm. 412, 115(2011).

5. Cahlin C., Lennroth C., Arvidsson A.: Int. J.Oncol. 32, 909 (2008).

6. Kismet K., Akay M.T., Abbasoglu O. ErcanA.,: Cancer Detect. Prev. 28, 127 (2004).

7. Tarnawski A.S., Jones M.K.: J. Mol. Med. 81,627 (2003).

8. Saikawa Y., Sugiura T., Toriumi F.: AnticancerRes. 24, 2723 (2004).

9. Ohtani H., Ikegawa T., Honda Y.: Pharm. Res.24, 1936 (2007).

10. Altorki N.K., Keresztes R.S., Port J.L.: J. Clin.Oncol. 21, 2645 (2003).

11. Naigang G.: Shangdong Med. J. 49, 75 (2009).12. Han C., Michalopoulos G.K., Wu T.: Cell

Physiol. 207, 261 (2006).

Received: 2. 08. 2015


Non-conventional formulations such as phar-maceutical drug delivery systems containing a deliv-ery base and an active agent have created new rulesfor the home use of medicine. Transdermal thera-peutic systems (TTS), a significant widespreadgroup of pharmaceutical dosage forms that enableeither systemic or topical drug absorption throughthe skin, are advantageous as they increase not onlytherapeutic effectiveness but also patient compli-ance, including:● The absorption of the drug independent of gas-

trointestinal absorption conditions,● No first-pass metabolism occurs during absorp-

tion through the skin, ● Prolonged release can be achieved, even for sev-

eral days,● Lower fluctuations in plasma concentrations,● Alternative solutions to the number of medicines

in polypharmacy, ● Alternative application route in swallowing diffi-

culties, dysphagia, and vomiting, and ● Potential alternatives to invasive interventions

reduce the risk of infections in a hospital setting (1).In addition, the dosing frequency of a particular

drug can be decreased with the application of patches,

in several therapeutic areas: the β2-agonist tulobuterolpatch demonstrated a significantly higher compliancerate than the corresponding inhalation therapy in asth-ma or COPD treatment (2), or, in the case ofParkinsonís disease, the rotigotine patch (3), and hor-monal contraceptives are preferred in patch rather thanoral dosage form (4). Alternatively, patient compli-ance can often be increased by a decrease in theunpleasant side effects of certain active substances.The reason for this phenomenon may be lower andmore uniform plasma concentrations or the avoidanceof the gastrointestinal tract and the first-pass effect.

Pharmaceutical care as a documented activityof pharmacists plays an increasing role in manyEuropean countries in the 21st century (5, 6).Hungarian pharmacies strive to provide apprentice-ships in appropriate drug therapy based on a patient-centered care model aimed primarily at improvingpatient quality of life in a world of product-orientedpharmaceutical companies (5). This professionaltask includes three areas: ● Medication therapy management,● Cooperation with the doctors who are prescribing

medications, and● Patient education and consultation (6).

GENERAL

PATIENTSí KNOWLEDGE OF MEDICAL PATCHES IN HUNGARY

ORSOLYA SOMOGYI and ROM¡NA ZELK”*

University Pharmacy, Department of Pharmacy Administration, Semmelweis University,H-1092 Budapest, , Hıgyes E. Street 7-9., Hungary

Abstract: Transdermal therapy with medical patches is a simple possibility in home medication. As the correctuse of patches has a decisive impact from the point of its modulator effect.A questionnaire survey was devel-oped to explore level of patientsí knowledge of the correct use of transdermal patches. A survey was adminis-tered in thirteen Hungarian community pharmacies from October of 2012 to May of 2015. Most of the partici-pants, men and women over 18 years of age (n = 233), used major analgesic patches (fentanyl); the remainderwere given nitroglycerin, NSAID analgesics patches during the survey. For the hypothesis testing it wasassumed that men were more likely to use a razor for skin depilation before patch application than women astheir denser pelage hinders patch adhesion. The hypothesis testing showed no significant gender difference inrazor use (χ2 = 0.201; p = 0.654). Pharmacists should direct patients to avoid using soap for skin cleansingbefore patch application because only 22 percent of the participants always avoided its use. Since only 9 testswere flawless from 233 completed questionnaires. Many patients do not understand how to correctly apply atransdermal dosage patch. Pharmacists should teach their correct application based on results.

Keywords: medical patch, counseling, compliance, application, instruction, survey

1653

* Corresponding author: e-mail: [email protected]; phone: +36-12170927

1654 ORSOLYA SOMOGYI and ROM¡NA ZELK”

An improvement in patient adherence to drugtherapy has become essential because more andmore patients are forced to continually take morethan one drug at a time (7, 8).

National protocols and working models haveboth been developed to improve pharmaceuticalcare and consultation, which is an increasinglydeveloping area of pharmacy practice in Hungary(5, 6, 8-12).

The purpose of the present study was to obtaininformation from a short questionnaire survey aboutpatient knowledge (without counseling) of the regu-lar or occasional application of patches. The surveyprovides a sense of what kind of information trans-fer would improve self-medication administrationand increase patient compliance in the use of trans-dermal patches. In the course of the survey eachpatient received a brochure containing the correctanswers. (Appendix 1). Based on our pharmacyísexperience, patients can correctly answer to theproblematic questions during dispensing medicalpatches after they read the information brochure. Inthe future we would like to measure this improvingeffect of this brochure and find a proof for that.

Each pharmacy signed a declaration confirm-ing the regular procedure. As each patient voluntar-ily participated in the survey, which is a non-inter-ventional study, therefore no further ethicalapproval was required.

METHOD

Patient questionnaire survey

The survey was administered in thirteen com-munity pharmacies of different regions of Hungary(Central Hungary: Duna˙jv·ros, R·calm·s, Ercsi,Csob·nka; West Hungary: Celldˆmˆlk, Kapuv·r;North-West Hungary: Tata; North Hungary:SzikszÛ; South Hungary: Kajdacs, Kˆlesd,

S·rszentlırinc and Budapest) from October of 2012to May of 2015. The pharmacies were a varied oflow, medium and high traffic community pharma-cies. Patients were surveyed personally by pharma-cists and assistants after dispensing the medicalpatches without counseling and helping. Answeringthe single-page questionnaire took about five to tenminutes and had to be completed directly after thepurchase of the patches. The questionnaire includedquestions suitable for the exploration of applicationerror frequency; it was important that the correctanswer not be obvious. It contained six closed-typequestions, the answers to which had to be chosenfrom a five-point scale. These closed questionsenabled simple and quick responses and reduceddiscrimination against those who were less literate,thus assuring a standard manner of administrationwithout influencing the patients. Moreover, it waseasy to code, record, and analyze the results quanti-tatively.

Response options and their evaluation

Patients used a five-point scale to denote theirresponses in the following categories:

1) I never use it in this way 2) I rarely use it in this way3) I sometimes use it in this way4) I usually use it in this way 5) I always use it in this wayThis five-point scale was transformed into a

two-point scale to obtain a more parsimoniousmeasure by which to evaluate the influence of age,gender, and education on the incorrect use of trans-dermal patches. Another consideration was to obtaina sufficient sample size for statistical testing (vari-able frequency).

This means in practice that the ìclearly cor-rectî answer is selected in each of the six questionsand all other answers grouped into a new ìwrongî

Table 1, "Clearly correct" answers for Questions 1 to 6.

Questions "Clearly correct" answers

Q1: I use a razor for skin depilation before applying the patch. 1) I never use it in this way (8)

Q2: I cleanse the affected skin with soap or shower gel 1) I never use it in this way (8, 9)thoroughly before applying the patch.

Q3: I apply the patch directly after hydrating my skin with body lotion. 1) I never use it in this way (8, 9)

Q4: I try to apply every new patch to a different 5) I always use it in this way (8, 9)location on my body (even on the affected body part)

Q5: I apply the patch in the middle of my back because it is 1) I never use it in this way (8-10) less exposed there, as exposure may damage the patch.

Q6: I am aware that the patch should adhere tightly to my skin. 5) I always use it in this way (8-10)

Patients' knowledge of medical patches in Hungary 1655

answer category. Table 1 (13-15) presents theìclearly correctî answers for Questions 1 to 6.


IBM SPSS statistics was used. For hypothesistesting and the more other descriptive statistical testsχ2 tests were selected. Fischer exact test was usedaccording geographic settlements analysis.

Hypothesis testing (Q1)Men may be more likely to use a razor for skin

depilation before applying a patch than womenbecause the denser pelage on their shoulders, legs,or waist can decrease patch adhesion to the skin. Thehypothesis testing was completed with a χ2-test (α =0.05).

Additional statistical testsIn processing the data more statistical analyses

were made with a χ2-test (α = 0.05) for the distribu-tion of ìwrongî answers (Q1ñQ6). It showed themost frequent application error and any significantdifference between the incidences of these errors.

The distribution of ìwrongî answers wasexamined for each question for the analysis of theeffect of education and age. The analysis of ìwrongîanswers (Q1ñQ6) by education level was not per-formed with a χ2-test according to the question-naireís six education levels because the incidencewas less than 5 percent in over 20 percent of thecells. Therefore, we developed three groups: lowereducation (general and vocational training school),secondary education (secondary school and high

school), and higher education (college and universi-ty). Based on this grouping, it became apparentwhich application problems pharmacists shouldfocus on regarding patientsí gender and education.

The analysis according to geographic settle-ments was made with Fischer exact test because theincidence was low in cells to χ2-test.

RESULTS

Description of the sample

233 patients participated in the survey whowere women aged 18 and over (n = 153) and menaged 18 and over (n = 80). They placed themselvesin one of four age groups: 18ñ30 years (n = 32),31ñ50 years (n = 90), 51ñ70 years (n = 76), and 71years and older (n = 35). They also had to choosetheir level of education from six categories: primaryeducation (n = 28), vocational school graduates (n =28), secondary school graduates (n = 50), highschool graduates (n = 47), college graduates (n =47), and university graduates (n = 33). As the focuswas the use of patches, those regularly using fen-tanyl, nitroglycerin, or NSAID-containing analgesicpatches.

Result of hypothesis testing

The distribution of responses to Question 1, ìIuse a razor for skin depilation before applying thepatch,î by gender: only 59.5 percent of females and62.5 percent of males selected the correct option ofìnever.î This means that a considerably high ratio ofpatients use a razor before applying their patches;

Appendix 1. Patient information leaflet for the appropriate use of transdermal patches (13-15).

● Before applying a transdermal patch you should never use a razor for skin depilation because a razor can causesmall lesions on your skin's surface, which might affect the absorption of the drug. If necessary, use scissors forskin depilation (13).

● The patch should be applied to clean, dry skin. You may cleanse the skin but use only clean water and thenwipe the skin, because any chemicals can modify the release and liberation of the active substance from thepatch (13, 14).

● Avoid using oils and creams before applying the patch because they too can damage the effect of the drug (13,14).

● The location of patch application should be alternated. If you would like to use a patch on the same skin areaagain, you must let the affected surface be patch-free for at least 3 days (13, 14).

● In chronic illness therapies, it is not necessary to place the patch in the middle of your back because it is diffi-cult to affix elsewhere on the body; you should look for a place on your body where you can see the patch welland do not forget to change the location, for example, shoulders, scapulas, abdominal wall, etc. (13-15). (Apatch can be used on a child's back because he or she cannot take it off easily and parents supervise the applica-tion.)

● In applying a patch, ensure that it adheres tightly to the surface of your skin. Avoid the body's bights (knees,waist, underarm, elbows, etc.). Exert pressure on the patch on your skin for 30 seconds (13-15).

1656 ORSOLYA SOMOGYI and ROM¡NA ZELK”

this could modify the absorption of the drug fromthese patches (13). Hypothesis testing did not showa significant difference between the frequencies ofrazor use by gender (χ2 = 0.201; p = 0.654).

Descriptive statistics

Table 2 illustrates the percent distribution ofresponses to Questions 2 ñ 6.

In the case of Question 2, only 21.9 percentanswered ìclearly correctî; this means that theremaining 78.1 percent used soap frequently tocleanse the skin before applying the patches and35.6 percent selected ìalways,î which was obvious-ly the worst choice.

The percent distribution of responses toQuestion 3-the frequency of body lotion use beforeapplying a patch-is higher than that for Question 2;this indicates that the ìclearly correctî answer rate ishigh. 72.5 percent ìneverî use body lotion beforeapplying a patch; only 3.9 percent ìalwaysî uselotion, and selected the worst answer option.

In Question 4, 30 percent selected the ìclearlycorrectî answer, that of ìalwaysî trying to applyeach new patch to a different location; 27.9 percentof the responses were acceptable: the ìusuallyî cat-egory. The distribution of the other 42.1 percent isvirtually equable: 15.5 percent selected ìnever,î 9.4percent ìrarely,î and 17.2 percent ìsometimes.î

In Question 5, a high ratio selected ìclearlycorrectî: 55.4 percent. Here it is assumed that thepatch is affixed to a highly visible, easily accessiblelocation on the body.

Finally, 70.8 percent selected the ìclearly cor-rectî answer to Question 6, that of ìalwaysî adher-ing the patch tightly to their skin, but the 5.2 percentwho answered ìneverî do not pay attention to itscorrect application. This is a serious mistakebecause inadequate patch adhesion damages theeffectiveness of their therapy (13, 14)

The distribution of ìwrongî answers means48.15 percent were from all responses to Question 1to 6. The difference was significant, according to the

number of ìwrongî answers: χ2 = 192.075 and p <0.001 in the six questions.

The questions with the highest number oferrors (ìwrongî answer category) were 2 (78.11%),4 (70.0%), 5 (44.63%), and 1 (39.48%) but low incase of 3 (27.5%) and 6 (29.18%).

Each of the six answers was selected ìclearlycorrectî without error (without ìwrongî answer) by9 patients from 233 participants and 13 patientsselected the ìwrongî answers on all six questions.32 patients answered with 1 error, 63 patientsanswered with 2 errors, 50 patients answered with 3errors, 43 patients answered with 4 errors and 23participants answered with 5 ìwrongî answers in thequestionnaire survey.

Effects of education level and age on the results

The only question with a significant differenceamong the three educational level groups of patientswas Question 6: p = 0.036. Also Question 6 waswith significant difference among the four age-groups of patients (p = 0.050).

Effects of settlementsí various on the results

Significant difference was according geo-graphic settlements in answers of Question 1, 2 and3. (Q1: p = 0.009; Q2: p = 0.023; Q3: p = 0.005).


Based on this questionnaire survey pharmacistsshould inform patients about the most frequentapplication errors, alerting them to avoid the use ofsoap for skin cleansing before a patch is affixed (themost frequently error) and that every patch must beaffixed to a different location on their body, if pos-sible (the second frequently error).

A considerably high ratio of patients use arazor before applying their patches; this could mod-ify the absorption of the drug from these patches(13). Women should also be cautioned against theuse of razors before affixing a patch. This problem

Table 2. Ratio of all responses in various questions (Q2 - Q6).

Responses Responses Responses Responses Responses Q2 (%) Q3 (%) Q4 (%) Q5 (%) Q6 (%)

Always 35.6 3.9 30.0 5.2 70.8

Usually 21.5 3.0 27.9 6.9 19.3

Sometimes 8.2 5.2 17.2 12.4 2.6

Rarely 12.9 15.6 9.4 20.2 2.1

Never 21.9 72.5 15.5 55.4 5.2

Σ: 100 100 100 100 100

Patients' knowledge of medical patches in Hungary 1657

was not assumed, but the results show the opposite. Most patients ìneverî use body lotion before apply-ing a patch, it is positive result. High ratio of partic-ipants sticks a patch to a highly visible, easily acces-sible location on their body. That does not seem animportant application error.

The ìnot tightly adhering to the skinî is a seri-ous mistake because inadequate patch adhesiondamages the effectiveness of their therapy (13-15),but this mistake is committed by only a few patients.Primarily, over 30 years old and secondary educa-tion or lower education patients should be moreaware of sticking the patch tightly to adhere theirskin.

Patients responded (Q1-Q3) better in the phar-macy of small settlements (e.g., Kajdacs, Csob·nka)than in high traffic pharmacies (e.g., Budapest).

It is interesting to compare the frequency ofsoap usage (more frequently) contra the frequencyof applying body lotion (less frequently), becauseboth decrease the effectiveness of the therapy (13,14). In practice pharmacists should compare theeffect of soap to body lotion usage.

The better responses in the small settlements(e.g., Kajdacs, Csob·nka) could be that the pharma-cists have more time for a patient in these small, lowtraffic pharmacies.

The results of the questionnaire survey showthat patients are unaware of even simple rules for theapplication of transdermal patches. Since only 9tests were flawless from 233 completed question-naires it shows the relevance of theme.

Acknowledgments

The authors are grateful to the Patika 52Pharmacy, Patika Libra Pharmacy, KiscelliPharmacy, Celli Szent M·rton Pharmacy, br. EˆtˆvsJÛzsef Pharmacy, GonviselÈs Pharmacy, Zˆldke-reszt Pharmacy, Margit Pharmacy, Dr. LumniczerS·ndor Pharmacy, Capricornus Pharmacy, Andr·ssy

Pharmacy, Szent MiklÛs Pharmacy, T&E Patik·dPharmacy for their valuable contribution in the sur-vey.

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Received: 24. 11. 2015


Tuberculosis (TB) is a contagious diseasecaused by a bacterium, Mycobacterium tuberculosis.TB mainly molests lungs but severe infection canalso involve vital body organs such as kidney, spineand brain. Upon coughing or sneezing, the TBpatient expels small aerosol droplets in air that carryinfectious bacteria. In this way, a TB carrier cancontaminate the healthy surrounding and inducts hisfellows on the risk of being infected by TB (1).Every year, around 9 million new TB cases arereported and approximately 2 million of thesepatients die (2). As reported by stop TB partnershipin 2010, 85% TB cases were reported in Africa(30%) and Asia (85%) although per capita incidencewas found to be falling 1% per year and death ratehas reduced to one third since 1990 (3). Cost of TBtreatment is related to sex, age, stage of TB andinsurance coverage. If the annual income is consid-ered near 8000 $ in 2008, the expenditure of TBcould pose catastrophic financial constraints for anyfamily (4).

Pakistan is a developing country and TB hasposed a serious health challenge. Pakistan is ranked5th in the high TB burden countries and estimated tohave 4th highest prevalence of multidrug resistance(MDR) TB. In Pakistan, 420,000 new TB casesappear each year and mortality rate is 39 per100,000 (6). Most Pakistani population belong tolower middle class thus direct and indirect costsassociated with TB treatment may appears to be akey concern for them. In Pakistan, 75% TB patientshave age between 19 to 50 years who are mostlyresponsible for earning family livelihood (8). Thus,TB could have far-reaching economic and socialconsequences for the infected individuals, their fam-ilies and communities. The study of Ali et al.showed that majority of TB patients were poor andconsulted conventional health care professionals ini-tially. The correct diagnosis of TB was discoveredonly after repeated visits. This resulted in advance-ment of illness which tend to increase the cost oftreatment by many folds (9).

SOCIO-ECONOMIC CONSTRAINS FACED BY TB PATIENTS THAT LEAD TO NON-COMPLIANCE ñ A CROSS SECTIONAL STUDY IN SOUTHERN

PUNJAB, PAKISTAN

AUBID ULLAH KHAN, NAVEED AKHTAR, ASADULLAH MADNI*, NAYAB TAHIR, MUBASHAR REHMAN and AHMED RAZA

Department of Pharmacy, The Islamia University, Bahawalpur, Pakistan

Abstract: Tuberculosis (TB) is an infectious disease that is communicable from one person toanother. Pakistan stands forefront among few unfortunate countries that still have heavy burden ofTB infection. Being a developing country, TB patients in Pakistan have to face different socio-eco-nomic constraints that upset life of the patients as well as their families. A cross sectional surveywas conducted in three selected districts, Lodharan, Bahawalpur and Bahawalnagar during February2011 to June 2011. From three hundred selected patients 210 were enrolled in study after receivingwritten consents. Data were collected though structured questionnaire and verbal interviews and sta-tistically analyzed by using the univariate analysis. The survey results showed that the low educa-tional status (p < 0.0012, CI 95%), unawareness of disease (88.7%), crowded population (p =0.0000, CI, 95%), poverty, high treatment cost and distant access to public health facilities weredirectly related to prevalence of TB. Different disease related constraints including poor attitude offamily members, colleagues, society and even health care professionals (p = 0.0000, CI 95%) werealso found to be major social factors leading to non-compliance and denial of TB treatment. Socio-economic constraints such as low literacy rate, unemployment, unawareness of disease, high treat-ment cost, poor attitude of family, society and health care professionals were directly related tononcompliance and should be given high priority consideration for achieving better TB managementand mitigation.

Keywords: tuberculosis in Pakistan, non-compliance, socio-economic, social stigma

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* Corresponding author: e-mail: [email protected]; phone: +92-0300-6821979, fax: +92-62-9255565

1660 AUBID ULLAH KHAN et al.

Social contact with family, friends and rela-tives constitutes a very essential part of social life. InPakistan, people avoid TB patients due to fear ofgetting infection. The social life of TB patients isstigmatized and affected negatively due to poorknowledge of disease, different socio-cultural mythsand misconceptions in general public. In a study,around 57% respondents proposed that separatingdishes of TB patients could be a good preventivestrategy (10), while in another study, 76% respon-dents proposed that patients of TB should be treatedin hospitals or sanatoriums (11). Almost 40%respondents claimed that they would not marry aperson who had active TB or even history of TB inthe past. Due to all these fears, most TB patientskept their disease hidden from their friends, relativesor from both. This defiant behavior of the patient ledto denial of the treatment, represented by 50% to70% defaulting rate in different treatment regimensthat led to emergence of drug resistance and new TBcases (12, 13). Muynck et al. comprehensivelyreviewed Pakistan national TB control program(NTP) and concluded that NTP has not been imple-mented to required extent and may not be able tofulfill its objective (14, 15).

The economic impact of TB and fear of socialstigma tend to drive the patients away from seekingtreatment. An insight into different socio-economicconstrains may help to find the answer to the mostbitter question associated with TB; why patients ofsuch a horrible disease commit non-compliance?

Objective of the present study was to identifyand evaluate different socio-economic constraintsthat a TB patient has to face over the course of treat-ment. The study has also aimed to find the signifi-cance association among the different socio-eco-nomic variables and emergence of non-complianceduring the course of TB therapy.

Methodology

Cross sectional descriptive study design wasadopted to study socioeconomic factors of noncom-pliance in TB patients from February 2011 to June2011. Structured questionnaire and verbal interviewwere used as most suitable data collection toolbecause of unavailability of complete medicalrecord and patientís history in the selected clinicalsettings. Sample size for the present study was cal-culated based on disease prevalence in the studyarea (15). In the present study, 300 TB patients werecontacted by using the convenience sampling tech-nique from the TB population registered underprovincial tuberculosis program in Bahawalnagar,Lodharan and Bahawalpur districts of Southern

Punjab. Only 210 patients provide written consentfor survey and were enrolled in the study. These dis-tricts were selected due to high prevalence of, andbeing indicated as hotspot of tuberculosis by nation-al tuberculosis program. The survey questionnaireinquired information about; 1. previous disease his-tory; 2. family size; 3. position in family; 4. infor-mation of infection during treatment; 5. nature ofdependents; 6. source and extent of monthlyincome; 7. family attitude/behavior to the patients;8). attitude of close friends/colleagues/health physi-cian to the patient; 9. support or motivation fromgovernment or any NGOs; 10. change of mood afterstart of medication; 11. acquisition of TB drugs andprocesses of the TB treatment; 12. prevalence ofcommon adverse effects; 13. access to TB HealthCare Centers; 14. constraints in drugs receivingprocess; and 15. quantity of available drugs fromhealth Centers. Data entry was done by using MSExcel 2007 and analyzed with SPSS version 10.0.Means and standard deviation (SD) were computedfor the continuous variables i.e., age and treatmentduration before noncompliance. The independentassociation and important risk factors were deter-mined using the univariate regression analysis bytaking p-value ≤ 0.05 as statistically significant witha confidence interval (CI) of 95%.

RESULTS

The survey involved active TB patients withthe aim to identify and evaluate different socio-eco-nomic constraints that TB patients had to face inSouthern Punjab, Pakistan. Demographic character-istics and their statistical significance are shown inTable 1. No significant difference was found in gen-der (p > 0.05) as both males and females had similarprevalence. Difference in age groups, marital status,treatment duration before noncompliance and litera-cy rate were found to be highly significant (p <0.0012). Present study indicated that TB awarenesswas very poor in general public as well as patients.Most of the patients believed that TB is a contagiousand almost half of them correctly described thesymptoms of the TB. However, 88.7% patients werenot familiar with the risk factors and the precautionsthat should be adopted during the course of thetreatment.

With respect to economic status, we found that51% patients had more than 8 family members, 40%patients had 4-8 family members, 8% patients had 2family members while only one patients (0.5 %) hadone family member (p = 0.0000, CI, 95%). A major-ity of TB patients (92%) were heads of the family

Socio-economic constrains faced by tuberculosis patients that lead to... 1661

and responsible for all the liabilities of their fami-lies. Of the 193 head of families, 52% patients hadmore than 8 dependents. In this study, 203 (96.7%)patients claimed that they were already sufferingfrom miserable economic conditions before con-tracting TB. Results regarding employment status ofTB patients showed that 75.3% patients were unem-ployed (Fig. 1). Of the remaining 23.7% employedpatients, 17.3% had monthly income less than 80USD, 3.3% had monthly income less than 150 USDand only 2.9% had monthly income more than 150USD and less than 300 USD (p = 0.0000, CI 95%).Results of this study also identified delayed diagno-sis as key factor; almost 44.3% patients were diag-nosed with TB after 1 year while 33.9% patientstook more than 2 years to be diagnosed properly.

With regards to social status, all TB patientscomplained that they have been facing bad to worstbehavior of the society (Fig. 2). Patients (58%) com-plained that the behavior of their family turned toworst after they were identified with TB (p =0.0000, CI 95%). When questioned about behavior

of society, 59% patients complained worst and hate-ful behavior of their colleagues (p = 0.0000). Fromhealth care providers perspective, results showedthat 41.7% patients complained worst behavior ofphysicians and health care workers (p = 0.0090. CI95%). In addition, 92.9% patients under studyresponded that they neither received any motivationfrom the society nor any support from governmentsand non-government organizations. Most patientsreported change in their attitude with the disease;92.3% patients felt that they had developed differenttypes of feelings and attitude (p = 0.0045).

Another important aspect was patients reportedproblems with drugs and drug seeking process. Mostpatients reported that drugs were unbearable (90%),partially available (82%) and difficult treatment pro-cedure (73%). Patients reported high prevalence ofcommon adverse effects of TB drugs such as insom-nia (62.9%), nausea (22.8%) and headache (14.3%).Distance to the nearest health care facility was alsofound to be a treatment limiting factor in 74%patients.

Table 1. Analysis of different demographical variables of study participants.

Number of Variables patients Mean Standard p value*

(percentage) deviation

Gender

Male 144 (68.6%) - -0.0812

Female 64 (31.4%) - -

Age

< 20 years 16 (7.6%) 15.9357 2.3840

20-40 years 110 (52.4%) 31.1818 5.3499

40-60 years 55 (26.2%) 28.8727 5.15240.0012

> 60 years 29 (13.8 %) 72.0000 7.5155

Marital status

Married 195 (92.9%) - -0.0000

Unmarried 15 (7.1%) - -

Treatment duration before noncompliance

< 1 month 23 (11%) 0.688 0.1927

< 2 months 46 (21.9%) 1.6 0.2263

< 3 months 101 (48.1%) 2.5495 0.27590.0000

4 months or more 40 (19 %) 3.4925 0.2473

Education status

Illiterate 171 (81.4%) - -

Primary 34 (16.2%) - - 0.0000

Matriculation 5 (2.4%) - -

Sample size consisted of 210 noncompliant TB patients; * result is statistically significant when p ≤ 0.005, CI 95%


DISCUSSION

Pakistan is a populous country and most of itspopulation (almost 68%) resides in rural areas thatlack sufficient health care facilities. SouthernPunjab areas of Pakistan mainly consists of villagesand underdeveloped areas with inadequate educa-tion. The study in the three different hospitals ofGreek indicate that the non-compliance was con-tributed by the low literacy in 71.80% patients (16)that strengthen the results of present study. Due tolow literacy rate the patients just knew the signs andsymptoms of disease, but were unaware of different

risk factors and precautions that should be adoptedduring the course of the treatment. This lack ofawareness lead to poor control of disease and itsspread to the society (10). These findings are inaccordance with the results of Rashmi et al. whichfind that majority of the patients in Mysuru wereunaware of the contagious nature and other risk fac-tors associated with the transmission of the disease(17). The study results also demonstrated that amajority of patients had populous families and didnot care if they were spreading disease to others.This was a very alarming situation in terms of safe-ty. According to World Health Organization, one

Figure 1. Employment status and monthly income (USD per month) of TB patients

Figure 2. Behavior of family, colleagues and health physicians towards TB patients

Socio-economic constrains faced by tuberculosis patients that lead to... 1663

untreated TB patient may spread the disease to 10-15 healthy persons including those who are takingcare of TB patients and their children (18). Variousstudies in Pakistan and India showed that healthworkers and media are the major source of informa-tion regarding health related problems (19, 20).Thus, health care centers should be established ded-icated information sections that provide informationpolitely on various risk factors, precautions andtreatment associated complication in order to devel-op a comprehensive control strategy.

Social life and behavior have been correlatedwith prevalence of TB and its effects were estimat-ed in different studies. We found that more than halfof the patients reported bad to worst behavior fromfamily and colleagues alike. This behavior of haterresulted in loss of confidence and self-worth occa-sioned by rejection from people who were veryimportant to them (21). Fox et al. found that the dis-criminative behavior of the family and society(47%, OR 1.55, adjusted 0.95-2.53) was a morecommon perception in the TB patients of Vietnam(22). In addition, more than one third patients point-ed out that behavior of physician has been worst athealth care centers. Many patients claimed that theystopped visiting health care centers because ofdepraved behavior of the society. Compliance withtreatment regimen cannot be ensured without physi-cianís belief of an obligation to facilitate potentialnoncompliance patients. A physicianís role is topolitely make the patients informed of benefits ofcompliance and aware of the risk factors for nonad-herence. Any feeling of hater or avoidance wouldmake adherence more difficult to patients than it hasto be (12). These results supported the need ofimplementation of National TB Control Program(NTP) guidelines which urge physicians and healthcare workers to develop, alter or modify their behav-ior in order to motivate TB patients and ensurepatients compliance with treatment regimen (23).

Present study indicates that economic con-straints play a major role in emergence of non-com-pliance and associated denial of treatment. Mostpatients were poor. Patients initially spend heftysums on treatment from conventional under-quali-fied general health practitioners and conventionalmedicine practitioners. When TB was diagnosed,patients had to face various socio-economic con-straints including unemployment. Many patientsalso claimed that they had to face cut-off in workinghours due to disease. This makes patients tempted tohide the disease from colleagues and employers.These results also confirm the status of TB as ìthedisease of poorî. In Pakistan, a patient averagely

pays direct cost of PKR 1500-1800 per month forTB treatments (24). Unfortunately, a majority ofpatients were earning less than this. These econom-ic factors may ultimately lead to malnutrition, whichis itself a risk factor of non-compliance (24).Conditions could be especially awful for womenwho are dependents on their family for food andmedicines. TB infection in girls was found to beassociated with denial of marriages. Some marriedwomen also told that they were separated from theirchildren due to fear of spreading infection to chil-dren and other family members. In Southern Punjab,there were no effective financial aid schemes fromgovernment or non-government organizations(NGOs) and such families were mostly supported bythe kinships network (termed as Beradri in Urdu).Such support, if luckily available, could also help inincreasing patient compliance or adherence withtreatment (25).

All patients reported change in behaviorincluding different forms of anxiety or depression.Such mental stresses have been previously reportedas predisposing factor to infectious diseases such asTB (26). Common adverse effects of TB drugs werealso prevalent in all patients. Overall, TB drugs wereunbearable for most patients who tend to quit thetreatment for some days, or forever. Shameer et al.endorse the finding of this study that adverse drugreactions (OR 2.46, CI 95%) results in the noncom-pliance and interruption of the therapy (27). Scarcityof TB treatment centers and complexity of treatmentseeking process were also identified to be key factorfor delayed diagnosis and non-compliance withtreatment regimen. Herrero et al. (28) explain thattravelling cost (OR 2.5; CL 95%) may increase thefinancial burden on the patients that already facingthe high therapy cost. In many cases, patients had toborrow money to pay the high travel costs to andfrom the treatment centers. Drugs were partiallyavailable and patients had to wait up to 4 h to receivedrug. All these factors can stop treatment processpartially or completely for a period of time. If con-tinuously occurring, the issues related to availabilityof drugs and treatment process may lead to emer-gence of noncompliance (29). WHO (25) as well asStop TB Partnership (26), a global collaboration ofTB health workers community, have hailed recentefforts of national and provincial TB control pro-grams but also stressed that such programs need tobe speed up and expanded in order to envision com-plete eradication of disease. Investment in healthsector to effectively control TB will not only preventloss of money in term of treatment expenses but alsogenerates revenue due to improved health (30). Such


big segment of population can participate actively inthe development of society rather than being a bur-den on them (31).

CONCLUSIONS

Life of TB patients was stigmatized due to mis-erable economic conditions as well as bad attitude offamily and colleagues. Loss of job, malnutrition,highly populous housing and treatment costs werethe distressful economic constraints confronted byTB patients. Lack of knowledge, poor attitude ofsociety and predominance of myths and misconcep-tions about the disease also accounted for the emer-gence of non-compliance. Health care centers mustensure availability of TB drugs and dedicatedpatients education sections for complete eradicationof TB from Southern Punjab, Pakistan.

REFERENCES

1. Kumar V., Abbass A.K., Nelson F., Richard M.:Robbins Basic Pathology. 8th edn. p. 516.Elsevier Health Sciences, Philadelphia 2007.

2. Global tuberculosis control: World HealthOrganization, Geneva 2010. (WHO/HTM/TB/2010.7).

3. Stop TB Partnership. The Global Plan To StopTB 2011-2015. [updated 5 Mar 2010; cited 10thJune 2013].

4. World Development Report. WDR 1993:World Bank investing in health 1993.

5. Hossein Z., Mojgan A.: Eur. Resp. Soc. Anul.Cong. Vienna, Austria 2009.

6. Dennis A.A.: The Economic Impact ofTuberculosis ñ The Stop TB Series, MinisterialConference Amsterdam, March 22-24, 2000.

7. World Health Organization. World Health Sta-tistics - 2010. [cited 2013 Jun. 7]. Available from:http://www.who.int/whosis/whostat/ 2010/en/.

8. World Health Organization: WHO Report onthe tuberculosis epidemic. 1997 [updated 03Apr. 2003; cited 30th May 2013]. Availablefrom: https://scout.wisc.edu/archives/r745/

9. Gupta K.B., Gupta R., Atreja A., Verma M.,Vishvkarma S.: Lung India 26, 9 (2009).

10. Ali S.S., Rabbani F., Siddiqui U.N.: Int. J.Tuberc. Lung Dis. 7, 1052 (2003).

11. Khan J.A., Irfan M., Zaki A., Beg M., Rizvi N.:J. Pak. Med. Assoc. 56, 211 (2006).

12. Liefooghe R., Suetens C.H., Meulemans H.,Moran M.B., Muynck A.D.: Int. J. Tuberc.Lung Dis. 3, 1073 (1999).

13. Schmidt C.W.: Environ. Health Perspect. 116,478 (2008).

14. World Health Organization: World HealthReport: Primary Health Care (now more thanever). 2008. Available from: http://www.who.int/whr/2008/en/.

15. Muynck A.D., Siddiqi S., Ghaffar A., Sadiq H.:J. Pak. Med. Assoc. 51, 41 (2001).

16. Arapi K., Kioumis I., Ktenas E., Amanaki E.,Garani-Papadatos T.: Pneumon 28, 351 (2015).

17. Rashmi P., Williams S., Thomas U.M., Devi A.:Int. J. Nurs. Care 4, 27 (2016).

18. Naing, L., Winn T., Rusli B.N.: Arch. Orofac.Sci. 1, 9 (2006).

19. Renuka M., Dhar M.: J. Med. Sci. 5, 333(2012).

20. Ahmed A., Chaudhary G.A., Farooq H.: Med.Forum Mon. 25, 14 (2014).

21. World Health Organization.: Implementing theWHO Stop TB Strategy. WHO Publications,Geneva 2008.

22. Fox G.J., Nhung N.V., Loi N.T., Sy D.N.,Britton W.J., Marks G.B.: BMC Infect. Dis. 15,1 (2015).

23. Jacobson J.A.: Thorac. Surg. Clin. 15, 461(2005).

24. Farida H., Baig L.: J. Pak. Med. Assoc. 56, 207(2006).

25. WHO Report 2009: Global TuberculosisControl Annex 3: Epidemiology 2009 [cited2013 Jun 5].

26. Alavi H.: The Two Biradiris: Kinship in RuralWest Punjab. T.N. Madan, Ed., MuslimCommunities of South Asia: Culture, Societyand Power. New Delhi 2001.

27. Shameer K.M., Manjunath K., Sam D.M.,Krishnaveni V., Rakesh P.S., Jasmin H.P.: Int.J. Community Med Public Health, 3, 962(2016).

28. Herrero M.B., Ramos S., Arrossi, S.: Rev. Bras.Epidemiol. 18, 287 (2015).

29. Schneiderman N., Ironson G., Siegel S.D.: Ann.Rev. Clin. Psych. 1, 607 (2005).

30. Schulz R., Cook C., Roller L., Fincham J.,Gowan J.: Patient Compliance withMedications: Issues and Opportunities, CRCPress, London 2007.

31. Dulmen V.S., Sluijs E., DijkL V., de Ridder D.,Heerdink R., Bensing J.: BMC Health Serv.Res. 7, 55 (2007).

Received: 11.04. 2016


Male contraception is an interesting field ofresearch throughout the world. Even World HealthOrganization (WHO) formed coalitions withPakistan Government and international agencies tosupport research and activities in this field (1). Asafe and effective method for contraception with theleast side effects is a great need of today (2). Manyproducts are available in market mainly composedof toxic materials. Repeated use cause inflammationand increases risk of sexual transmitted diseases (3).Several chemical and biological methods have beenfailed in this regard and are considered injurious forhealth (4).

Nowaday's, phytomedicines are matter ofinterest for scientist for male contraception becauseof less side effects and cost effectiveness (5).Spermicidal effects of plants like Gossypiumherbaceum (6) Azadirachta indica (7), red algae G.acerosa (8) and Ruta graveolens (9) had been stud-ied by researchers. Present study was designed tocheck the effect of Hippophae rhamnoides berriesaqueous extract on human sperm motility by keep-ing in view the chemistry and folk use of plants ascontraceptives.

MATERIAL AND METHODS

Collection, authentication and pulverization

Hippophae rhamnoides L. berries were collectedfrom Cheema Laboratories, Civic Centre, TownshipLahore and dried under the shade at room temperaturefor about 5 days. The dried berries were pulverized toa fine powder and stored in amber colored bottles.

Preparation of water extractOne hundred fifty grams of dried crushed

berries were macerated in 1000 mL of distilledwater, for 48 h at room temperature and in a darkplace, and then filtered through a paper filter. Thewater from filtrate was removed in rotary vaccumevaporator. The reddish brown extract obtained waskept in clean jar at room temperature for furtherexperiments. About 30 g of the extract wasobtained.

Test for phenols Crude extract (0.1 g) was dissolved in 2 mL of

methanol and to this 2 mL of a 5% aqueous solutionof ferric chloride was added. A development of pur-ple coloration indicates the presence of phenols(10).

Collection of samples

Semen samples were obtained with masturba-tion, in diagnostic laboratory from ten patientsreporting at Andrology Department, FatimaMemorial Hospital, Shadman, Lahore All patientswere in between 35-45 year age. Written consentform was signed by each patient. Standardization ofabstinence time i.e., 3-4 days was maintained.Samples were collected in wide mouth, transparent,sterile containers. Samples with azoospermia orsevere oligozoospermia, sperm aggregation andsperm agglutination were excluded. The sampleswhich had sperm motility between 50 and 70%, 20◊ 106 spermatozoa/mL, 2.1 mL /ejaculate, pH 7.5-7.9, and with minimum contamination of debris or

SHORT COMMUNICATION

EVALUATION OF SPERMICIDAL PROPERTY OF HIPPOPHAE RHAMNOIDES L. BERRIES

SADIA CHUMAN1, HAMMAD SALEEM1*, M. SHOAIB ALI GILL1, NABEEL SHAHID, FIZZA KARAMAT1, TABISH ALI1, FAISAL NADEEM1 and WAQAS MEHMOOD2

1Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences, Lahore, Pakistan 2Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan

Keywords: contraceptive, Hippophae rhamnoides L. berries, spermicidal

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* Corresponding author: e-mail: [email protected]; phone: +92-3357885316

1666 SADIA CHUMAN et al.

cells other than spermatozoa were used for the assayin accordance with the World Health Organizationstandard.

Immobilization assay

Sperm count motility was assessed microscop-ically as per the WHO manual (11). The sampleswere placed in an incubator at 37OC for 25-30 min.Subsequently, the samples were well mixed for 20 sat 37OC. Different amounts of the extract (2, 5, 10,15 mg) were dissolved in 1 mL of physiologicalsaline (0.9% NaCl) after filtration through a 0.2 µmfilter; the filtrate was added to a semen sample in a1 : 1 v/v ratio. In the control group, physiologicalsaline (0.9% NaCl) was added to the same volumeof semen. Samples were mixed for 10 s and testedfor their effect on sperm motility. Ten µL of the mix-ture was immediately placed on a pre-warmed slideand 5 fields were viewed and at least 200 spermato-zoa were counted in duplicate, i.e., 400 spermatozoain total. At least five fields were assessed in eachcount. Within each field, all progressive spermato-zoa (WHO class a) and slowly progressive sperma-tozoa (WHO class b) were counted first. All cellswhich were present in the field were counted. If thenumber of progressively motile spermatozoa werehigh in the field, a smaller part of the field was used.When all progressive spermatozoa were countedthen non-progressive spermatozoa (WHO class c)and immotile spermatozoa (WHO class d) werecounted in the same field. The four categories wereexpressed as percentages (rapid, slow, non-progres-sive, and immotile). Duplicate counting was made todetect and minimize random errors. Assessment ofsperm motility was repeated on a second aliquot pre-pared in the same way. The averages for two countswere calculated and given as results. If the differ-ence between the two motility counts was too large,two new assessments were made. The lowest dosecausing 100% immobilization in aliquots was calledthe minimum effective dose (MED). The pH of con-trol and samples containing different concentrationsof extract was measured with pH strips.

Assessment of sperm viability and membrane

integrity

Sperm viability test was performed by eosin-nigrosin staining method (12) for MED treated sam-ples and that of the control group. Twenty five µL ofdye and the same amount of the test sample weremixed for 30 s. Then, a smear was obtained by a 15µL droplet of the mixture. After air drying the smearwas viewed microscopically. The percentages ofalive (white) and dead (red) cells were calculated by

at least counting 200 cells. As the sperms with intactmembrane do not undergo staining, this stainingshows the integrity of cell membrane too (13).

Statistical data analysis

All statistical calculations were performed withSPSS version 13 (Evaluation) for Windows. Theresults have been expressed as the mean ± S.E.%.The statistical difference among treatments groupsand control were evaluated by ANOVA (analysis ofvariance) followed by Tukey's HSD test. Mann-Whitney test was used to compare the two groups.The difference was consider significant when p <0.050.

RESULTS

Purple coloration was evident with ferric chlo-ride test, indicating the presence of phenolic com-pounds in the aqueous extract of Hippophae rham-noides berries.

After preliminary screening of extracts (petro-leum ether, chloroform, methanol), aqueous extractwas selected for the study due to its good results andcompatibility with semen samples. Different concen-trations of aqueous extract were tested as describedabove and immediate immobilizing effects of thematerial were seen in a dose-dependent manner. Theresults showed that aqueous extract has antimotilityactivity at all concentrations with respect to control.It was also observed that sperm motility decreasedwith an increase in dose of extract. Maximum inhibi-tion of sperm motility was observed at 15 mg/mLconcentration, where all sperms become immotileimmediately and was significantly different fromcontrol, 2 and 5 mg/mL concentrations (p = 0.000)but was insignificantly different from 10 mg/mL (p =0.056). Fifteen mg/mL concentration was chosen asthe minimum effective dose (MED) in other experi-ments. Sperm motility in 2 mg/mL was not signifi-cantly different (p = 0.719) from control group. Thetotal motile sperm counts were significantlydecreased in 5 mg/mL (p = 0.000) concentration,when compared with control and other concentra-tions (2, 10, 15 mg/mL). Sperm motility was alsoinhibited significantly in 10 mg/mL (p = 0.000) incomparison with control group, 2 and 5 mg/mL con-centrations but the total motile sperm count in thisconcentration was insignificantly different from 15mg/mL (p = 0.056) (Table 1).

It was also observed that pH gradually changedfrom alkaline to acidic. The lowest pH (6.5) wasobserved at concentration 15 mg/mL where spermbecome totally immotile. Sperms were actively

Evaluation of spermicidal property of Hippophae rhamnoides L. berries 1667

motile in control and 2 mg/mL concentration groupwhere pH was slightly alkaline (Table 1).

Eosin-nigrosin technique results had shown thatthe immobilized effect was with impairment in cellviability and losing membrane integrity. Treated cellswere totally immotile at higher concentrations andthere was a significant difference (p = 0.000) betweenviability of the two groups (control & MED) (Table 2)

DISCUSSION

In the present study, in vitro effects of aqueousextracts of Hippophae rhamnoides berries on humansperm motility were studied. These immobilizingeffects were studied for the first time on the humansperm. The results (Tables 1, 2) showed that aque-ous extract has maximum antimotility activity athigher concentration of 15 mg/mL; extract inhibitssperm motility immediately at this concentration.This might be due to strong interaction between

sperm membrane and components of the extractwhich results in loss of membrane integrity andsperm viability. This was confirmed by eosin-nigrosin staining method (Table 2).

But still it is not known whether the immobi-lized effect was due to non-specific perturbation ofplasma membrane (14) or due to decreased and dis-turbed functions of plasma membrane enzymes likeCa2+ ATPase (15) and Na-K-ATPase activity (16) oralternatively it could have resulted due to detergentaction mediated by steroidal components i.e., sitos-terol, isofucosterol, campsterol, stigmastanol, cit-rostadienol, avenasterol, cycloartenol, 24-ethyl-enecycloartanol and obtusifoliol which are reportedto be present in berries (17), because Zia-Ul-Haq(18) reported that abridine (steroidal componentpresent in Abrus precatorius) interact with spermmembrane by this mechanism.

Development of purple color with ferric chlo-ride showed the presence of phenolic compounds in

Table 1. Total motile sperm count percentage in control and groups treated with different concentrations ofaqueous extract of Hippophae rhamnoides berries.

Total motileCondition sperm count %

pHDifference

(Mean ± S.E.)(p value)

(1-2) 0.719Control (1-3) 0.000

(1)64.00 ± 2.66 7.9

(1-4) 0.000(1-5) 0.000

(2-1) 0.7192 mg/mL (2-3) 0.000

(2)60.40 ± 2.38 7.9

(2-4) 0.000(2-5) 0.000

(3-) 0.0005 mg/mL (3-2) 0.000

(3)30.80 ± 2.61 7.5

(3-4) 0.000(3-5) 0.000

(4-1) 0.00010 mg/mL (4-2) 0.000

(4)8.00 ± 0.94 7

(4-3) 0.000(4-5) 0.056

(5-1) 0.00015 mg/mL (5-2) 0.000

(5)0.000 6.5

(5-3) 0.000(5-4) 0.056

Table 2. Comparative effects of aqueous extract of Hippophae rhamnoides berries at minimum effective dose(MED) and control on sperm viability.

Percentage of 15 mg/mLviable sperm (MED)

Control p value

(mean ± S.E.) 38.03 ± 2.46 60.00 ±2.66 0.000

1668 SADIA CHUMAN et al.

the extract. Phenolic and polyphenolic constituentsof plants have a strong inhibitory effect on spermmotility with dose-response relationship (19).

A phenolic content ranges from 1.9-23.5 mg/gin Hippophae rhamnoides berries and leaves (20).Berries are rich source of polyphenolic compoundse.g., rutin, quercetin-3-O-galactoside, quercetin,myricetin, kaempferol and iso-rhamnetin. Phenolicacids include gallic, protecatechuic, p-coumaric, fer-ulic, p-hydroxybenzoic, catechin and ellagic acid.

Antimotility effect might be due to phenols andphenol containg compounds because these com-pounds stimulate lipid peroxidation and generatefree oxygen species (ROS) (21). High levels of ROSin semen reduced sperm motility in vitro and causedamage to sperm nuclear DNA (22), sperm mem-branes and proteins due to interference and alter-ations in signal transduction mechanisms (23).

Phenols also cause disruption of ATP supplyfor sperm movement (24). Their activity was foundto be dependent on the positions of hydroxyl groupsand modulated by the positions of methyl groups onthe benzene ring (21).

According to Carr et al. (25) low pH of theseminal fluid is sufficient to cause inhibition ofsperm motility. This is in compliance with resultsbecause lowest pH was observed at higher concen-tration (15 mg/mL) that might be the reason ofdeclining motility.

CONCLUSION

In conclusion, the results of the study stronglyindicate that aqueous extract possesses contracep-tive properties but further seminal and chemicalinvestigations are required to find out the exactmechanism and site of action of aqueous extract onhuman spermatozoa.

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Received: 30. 10. 2015

Erratum 1669

In the paper entitled:

LEVEL A IN VITRO-IN VIVO CORRELATION DEVELOPMENT AND VALIDATION FOR TRA-MADOL HYDROCHLORIDE FORMULATIONS

byYANGPING CAI1, YOUSHAN LI2, SHU LI3, TIAN GAO3, LU ZHANG3, ZHE YANG4, ZHENGFU FAN3,

CHUJIE BAI3

published in: Acta Polonie Pharmaceutica ñ Drug Reseaech, Vol. 73, issue 5, pp. 1333-1338 (2016), the cor-rect authors affiliation shoud be:

1Intensive Care Unit, 2Peripheral Vascular Surgery, 4Emergency Department, Beijing University of Chinese Medicine, Dongzhimen Hospital, 100700, Beijing, China

3Peking University Cancer Hospital, Beijing, China

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